Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems and methods

ABSTRACT

This disclosure provides various TcBuster transposases and transposons, systems, and methods of use.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.62/435,522, filed Dec. 16, 2016, which application is incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 2, 2018, isnamed 52741-702_201_SL.txt and is 89,774 bytes in size.

BACKGROUND OF THE INVENTION

Transposable genetic elements, also called transposons, are segments ofDNA that can be mobilized from one genomic location to another within asingle cell. Transposons can be divided into two major groups accordingto their mechanism of transposition: transposition can occur (1) viareverse transcription of an RNA intermediate for elements termedretrotransposons, and (2) via direct transposition of DNA flanked byterminal inverted repeats (TIRs) for DNA transposons. Active transposonsencode one or more proteins that are required for transposition. Thenatural active DNA transposons harbor a transposase enzyme gene.

DNA transposons in the hAT family are widespread in plants and animals.A number of active hAT transposon systems have been identified and foundto be functional, including but not limited to, the Hermes transposon,Ac transposon, hobo transposon, and the Tol2 transposon. The hAT familyis composed of two families that have been classified as the ACsubfamily and the Buster subfamily, based on the primary sequence oftheir transposases. Members of the hAT family belong to Class IItransposable elements. Class II mobile elements use a cut and pastemechanism of transposition. hAT elements share similar transposases,short terminal inverted repeats, and an eight base-pairs duplication ofgenomic target.

SUMMARY OF THE INVENTION

One aspect of the present disclosure provides a mutant TcBustertransposase, comprising an amino acid sequence at least 70% identical tofull-length SEQ ID NO: 1 and having one or more amino acid substitutionsthat increase a net charge at a neutral pH in comparison to SEQ IDNO: 1. In some embodiments, the mutant TcBuster transposase hasincreased transposition efficiency in comparison to a wild-type TcBustertransposase having amino acid sequence SEQ ID NO: 1.

Another aspect of the present disclosure provides a mutant TcBustertransposase, comprising an amino acid sequence at least 70% identical tofull-length SEQ ID NO: 1 and having one more amino acid substitutions ina DNA Binding and Oligomerization domain; an insertion domain; a Zn-BEDdomain; or a combination thereof. In some embodiments, the mutantTcBuster transposase has increased transposition efficiency incomparison to a wild-type TcBuster transposase having amino acidsequence SEQ ID NO: 1.

Yet another aspect of the present disclosure provides a mutant TcBustertransposase comprising an amino acid sequence at least 70% identical tofull-length SEQ ID NO: 1 and having one or more amino acid substitutionsfrom Table 1.

In some embodiments, a mutant TcBuster transposase comprises one or moreamino acid substitutions that increase a net charge at a neutral pHwithin or in proximity to a catalytic domain in comparison to SEQ IDNO: 1. In some embodiments, the mutant TcBuster transposase comprisesone or more amino acid substitutions that increase a net charge at aneutral pH in comparison to SEQ ID NO: 1, and the one or more aminoacids are located in proximity to D223, D289, or E589, when numbered inaccordance to SEQ ID NO: 1. In some embodiments, the proximity is adistance of about 80, 75, 70, 60, 50, 40, 30, 20, 10, or 5 amino acids.In some embodiments, the proximity is a distance of about 70 to 80 aminoacids.

In some embodiments, the amino acid sequence of the mutant TcBustertransposase is at least 80%, at least 90%, at least 95%, at least 98%,or at least 99% identical to full-length SEQ ID NO: 1.

In some embodiments, the one or more amino acid substitutions comprise asubstitution to a lysine or an arginine. In some embodiments, the one ormore amino acid substitutions comprise a substitution of an asparticacid or a glutamic acid to a neutral amino acid, a lysine or anarginine. In some embodiments, the mutant TcBuster transposase comprisesone or more amino acid substitutions from Table 4. In some embodiments,the mutant TcBuster transposase comprises one or more amino acidsubstitutions from Table 2. In some embodiments, the mutant TcBustertransposase comprises one or more amino acid substitutions from Table 3.In some embodiments, the mutant TcBuster transposase comprises aminoacid substitutions V377T, E469K, and D189A, when numbered in accordancewith SEQ ID NO: 1. In some embodiments, the mutant TcBuster transposasecomprises amino acid substitutions K573E and E578L, when numbered inaccordance with SEQ ID NO: 1. In some embodiments, the mutant TcBustertransposase comprises amino acid substitution I452K, when numbered inaccordance with SEQ ID NO: 1. In some embodiments, the mutant TcBustertransposase comprises amino acid substitution A358K, when numbered inaccordance with SEQ ID NO: 1. In some embodiments, the mutant TcBustertransposase comprises amino acid substitution V297K, when numbered inaccordance with SEQ ID NO: 1. In some embodiments, the mutant TcBustertransposase comprises amino acid substitution N85S, when numbered inaccordance with SEQ ID NO: 1. In some embodiments, the mutant TcBustertransposase comprises amino acid substitutions I452F, V377T, E469K, andD189A, when numbered in accordance with SEQ ID NO: 1. In someembodiments, the mutant TcBuster transposase comprises amino acidsubstitutions A358K, V377T, E469K, and D189A, when numbered inaccordance with SEQ ID NO: 1. In some embodiments, the mutant TcBustertransposase comprises amino acid substitutions V377T, E469K, D189A,K573E and E578L, when numbered in accordance with SEQ ID NO: 1.

In some embodiments, the transposition efficiency is measured by anassay that comprises introducing the mutant TcBuster transposase and aTcBuster transposon containing a reporter cargo cassette into apopulation of cells, and detecting transposition of the reporter cargocassette in genome of the population of cells.

Yet another aspect of the present disclosure provides a fusiontransposase comprising a TcBuster transposase sequence and a DNAsequence specific binding domain. In some embodiments, the TcBustertransposase sequence has at least 70% identity to full-length SEQ ID NO:1.

In some embodiments, the DNA sequence specific binding domain comprisesa TALE domain, zinc finger domain, AAV Rep DNA-binding domain, or anycombination thereof. In some embodiments, the DNA sequence specificbinding domain comprises a TALE domain.

In some embodiments, the TcBuster transposase sequence has at least 80%,at least 90%, at least 95%, at least 98%, or at least 99% identity tofull-length SEQ ID NO: 1. In some embodiments, the TcBuster transposasesequence comprises one or more amino acid substitutions that increase anet charge at a neutral pH in comparison to SEQ ID NO: 1. In someembodiments, the TcBuster transposase sequence comprises one or moreamino acid substitutions in a DNA Binding and Oligomerization domain; aninsertion domain; a Zn-BED domain; or a combination thereof. In someembodiments, the TcBuster transposase sequence comprises one or moreamino acid substitutions from Table 1. In some embodiments, the TcBustertransposase sequence has increased transposition efficiency incomparison to a wild-type TcBuster transposase having amino acidsequence SEQ ID NO: 1. In some embodiments, the TcBuster transposasesequence comprises one or more amino acid substitutions that increase anet charge at a neutral pH within or in proximity to a catalytic domainin comparison to SEQ ID NO: 1. In some embodiments, the TcBustertransposase sequence comprises one or more amino acid substitutions thatincrease a net charge at a neutral pH in comparison to SEQ ID NO: 1, andthe one or more amino acid substitutions are located in proximity toD223, D289, or E589, when numbered in accordance to SEQ ID NO: 1. Insome embodiments, the proximity is a distance of about 80, 75, 70, 60,50, 40, 30, 20, 10, or 5 amino acids. In some embodiments, the proximityis a distance of about 70 to 80 amino acids. In some embodiments, theTcBuster transposase sequence comprises one or more amino acidsubstitutions from Table 2. In some embodiments, the TcBustertransposase sequence comprises one or more amino acid substitutions fromTable 3. In some embodiments, the TcBuster transposase sequencecomprises amino acid substitutions V377T, E469K, and D189A, whennumbered in accordance with SEQ ID NO: 1. In some embodiments, theTcBuster transposase sequence comprises amino acid substitutions K573Eand E578L, when numbered in accordance with SEQ ID NO: 1. In someembodiments, the TcBuster transposase sequence comprises amino acidsubstitution I452K, when numbered in accordance with SEQ ID NO: 1. Insome embodiments, the TcBuster transposase sequence comprises amino acidsubstitution A358K, when numbered in accordance with SEQ ID NO: 1. Insome embodiments, the TcBuster transposase sequence comprises amino acidsubstitution V297K, when numbered in accordance with SEQ ID NO: 1. Insome embodiments, the TcBuster transposase sequence comprises amino acidsubstitution N85S, when numbered in accordance with SEQ ID NO: 1. Insome embodiments, the TcBuster transposase sequence comprises amino acidsubstitutions I452F, V377T, E469K, and D189A, when numbered inaccordance with SEQ ID NO: 1. In some embodiments, the TcBustertransposase sequence comprises amino acid substitutions A358K, V377T,E469K, and D189A, when numbered in accordance with SEQ ID NO: 1. In someembodiments, the TcBuster transposase sequence comprises amino acidsubstitutions V377T, E469K, D189A, K573E and E578L, when numbered inaccordance with SEQ ID NO: 1. In some embodiments, the TcBustertransposase sequence has 100% identity to full-length SEQ ID NO: 1.

In some embodiments of a fusion transposase, the TcBuster transposasesequence and the DNA sequence specific binding domain are separated by alinker. In some embodiments, the linker comprises at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 15, at least 20, or at least 50 amino acids. In someembodiments, the linker comprises SEQ ID NO: 9.

Yet another aspect of the present disclosure provides a polynucleotidethat codes for the mutant TcBuster transposase as described herein.

Yet another aspect of the present disclosure provides a polynucleotidethat codes for the fusion transposase as described herein.

In some embodiments, the polynucleotide comprises DNA that encodes themutant TcBuster transposase or the fusion transposase. In someembodiments, the polynucleotide comprises messenger RNA (mRNA) thatencodes the mutant TcBuster transposase or the fusion transposase. Insome embodiments, the mRNA is chemically modified. In some embodiments,the polynucleotide comprises nucleic acid sequence encoding for atransposon recognizable by the mutant TcBuster transposase or the fusiontransposase. In some embodiments, the polynucleotide is present in a DNAvector. In some embodiments, the DNA vector comprises a mini-circleplasmid.

Yet another aspect of the present disclosure provides a cell producingthe mutant TcBuster transposase or fusion transposase as describedherein. Yet another aspect of the present disclosure provides a cellcontaining the polynucleotide as described herein.

Yet another aspect of the present disclosure provides a methodcomprising: introducing into a cell the mutant TcBuster transposase asdescribed herein and a transposon recognizable by the mutant TcBustertransposase.

Yet another aspect of the present disclosure provides a methodcomprising: introducing into a cell the fusion transposase as describedherein and a transposon recognizable by the fusion transposase.

In some embodiments of a method, the introducing comprises contactingthe cell with a polynucleotide encoding the mutant TcBuster transposaseor the fusion transposase. In some embodiments, the polynucleotidecomprises DNA that encodes the mutant TcBuster transposase or the fusiontransposase. In some embodiments, the polynucleotide comprises messengerRNA (mRNA) that encodes the mutant TcBuster transposase or the fusiontransposase. In some embodiments, the mRNA is chemically modified.

In some embodiments of a method, the introducing comprises contactingthe cell with a DNA vector that contains the transposon. In someembodiments, the DNA vector comprises a mini-circle plasmid. In someembodiments, the introducing comprises contacting the cell with aplasmid vector that contains both the transposon and the polynucleotideencoding the mutant TcBuster transposase or the fusion transposase. Insome embodiments, the introducing comprises contacting the cell with themutant TcBuster transposase or the fusion transposase as a purifiedprotein.

In some embodiments of a method, the transposon comprises a cargocassette positioned between two inverted repeats. In some embodiments, aleft inverted repeat of the two inverted repeats comprises a sequencehaving at least 50%, at least 60%, at least 80%, at least 90%, at least95%, at least 98%, or at least 99% identity to SEQ ID NO: 3. In someembodiments, a left inverted repeat of the two inverted repeatscomprises SEQ ID NO: 3. In some embodiments, a right inverted repeat ofthe two inverted repeats comprises a sequence having at least 50%, atleast 60%, at least 80%, at least 90%, at least 95%, at least 98%, or atleast 99% identity to SEQ ID NO: 4. In some embodiments, a rightinverted repeat of the two inverted repeats comprises SEQ ID NO: 4. Insome embodiments, a left inverted repeat of the two inverted repeatscomprises a sequence having at least 50%, at least 60%, at least 80%, atleast 90%, at least 95%, at least 98%, or at least 99% identity to SEQID NO: 5. In some embodiments, a left inverted repeat of the twoinverted repeats comprises SEQ ID NO: 5. In some embodiments, a rightinverted repeat of the two inverted repeats comprises a sequence havingat least 50%, at least 60%, at least 80%, at least 90%, at least 95%, atleast 98%, or at least 99% identity to SEQ ID NO: 6. In someembodiments, a right inverted repeat of the two inverted repeatscomprises SEQ ID NO: 6. In some embodiments, the cargo cassettecomprises a promoter selected from the group consisting of: CMV, EFS,MND, EF1α, CAGCs, PGK, UBC, U6, H1, and Cumate. In some embodiments, thecargo cassette comprises a CMV promoter. In some embodiments, the cargocassette is present in a forward direction. In some embodiments, thecargo cassette is present in a reverse direction. In some embodiments,the cargo cassette comprises a transgene. In some embodiments, thetransgene codes for a protein selected from the group consisting of: acellular receptor, an immunological checkpoint protein, a cytokine, andany combination thereof. In some embodiments, the transgene codes for acellular receptor selected from the group consisting of: a T cellreceptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor(CAR), or any combination thereof. In some embodiments, the introducingcomprises transfecting the cell with the aid of electroporation,microinjection, calcium phosphate precipitation, cationic polymers,dendrimers, liposome, microprojectile bombardment, fugene, direct sonicloading, cell squeezing, optical transfection, protoplast fusion,impalefection, magnetofection, nucleofection, or any combinationthereof. In some embodiments, the introducing comprises electroporatingthe cell.

In some embodiments of a method, the cell is a primary cell isolatedfrom a subject. In some embodiments, the subject is a human. In someembodiments, the subject is a patient with a disease. In someembodiments, the subject has been diagnosed with cancer or tumor. Insome embodiments, the cell is isolated from blood of the subject. Insome embodiments, the cell comprises a primary immune cell. In someembodiments, the cell comprises a primary leukocyte. In someembodiments, the cell comprises a primary T cell. In some embodiments,the primary T cell comprises a gamma delta T cell, a helper T cell, amemory T cell, a natural killer T cell, an effector T cell, or anycombination thereof. In some embodiments, the primary immune cellcomprises a CD3+ cell. In some embodiments, the cell comprises a stemcell. In some embodiments, the stem cell is selected from the groupconsisting of: embryonic stem cell, hematopoietic stem cell, epidermalstem cell, epithelial stem cell, bronchoalveolar stem cell, mammary stemcell, mesenchymal stem cell, intestine stem cell, endothelial stem cell,neural stem cell, olfactory adult stem cell, neural crest stem cell,testicular cell, and any combination thereof. In some embodiments, thestem cell comprises induced pluripotent stem cell.

Yet another aspect of the present disclosure provides a method oftreatment, comprising: (a) introducing into a cell a transposon and themutant TcBuster transposase or the fusion transposase as describedherein, which recognize the transposon, thereby generating a geneticallymodified cell; (b) administering the genetically modified cell to apatient in need of the treatment. In some embodiments, the geneticallymodified cell comprises a transgene introduced by the transposon. Insome embodiments, the patient has been diagnosed with cancer or tumor.In some embodiments, the administering comprises transfusing thegenetically modified cell into blood vessels of the patient.

Yet another aspect of the present disclosure provides a system forgenome editing, comprising: the mutant TcBuster transposase or fusiontransposase as described herein, and a transposon recognizable by themutant TcBuster transposase or the fusion transposase.

Yet another aspect of the present disclosure provides a system forgenome editing, comprising: the polynucleotide encoding a mutantTcBuster transposase or fusion transposase as described herein, and atransposon recognizable by the mutant TcBuster transposase or the fusiontransposase.

In some embodiments of a system, the polynucleotide comprises DNA thatencodes the mutant TcBuster transposase or the fusion transposase. Insome embodiments, the polynucleotide comprises messenger RNA (mRNA) thatencodes the mutant TcBuster transposase or the fusion transposase. Insome embodiments, the mRNA is chemically modified. In some embodiments,the transposon is present in a DNA vector. In some embodiments, the DNAvector comprises a mini-circle plasmid. In some embodiments, thepolynucleotide and the transposon are present in a same plasmid. In someembodiments, the transposon comprises a cargo cassette positionedbetween two inverted repeats. In some embodiments, a left invertedrepeat of the two inverted repeats comprises a sequence having at least50%, at least 60%, at least 80%, at least 90%, at least 95%, at least98%, or at least 99% identity to SEQ ID NO: 3. In some embodiments, aleft inverted repeat of the two inverted repeats comprises SEQ ID NO: 3.In some embodiments, a right inverted repeat of the two inverted repeatscomprises a sequence having at least 50%, at least 60%, at least 80%, atleast 90%, at least 95%, at least 98%, or at least 99% identity to SEQID NO: 4. In some embodiments, a right inverted repeat of the twoinverted repeats comprises SEQ ID NO: 4. In some embodiments, a leftinverted repeat of the two inverted repeats comprises a sequence havingat least 50%, at least 60%, at least 80%, at least 90%, at least 95%, atleast 98%, or at least 99% identity to SEQ ID NO: 5. In someembodiments, a left inverted repeat of the two inverted repeatscomprises SEQ ID NO: 5. In some embodiments, a right inverted repeat ofthe two inverted repeats comprises a sequence having at least 50%, atleast 60%, at least 80%, at least 90%, at least 95%, at least 98%, or atleast 99% identity to SEQ ID NO: 6. In some embodiments, a rightinverted repeat of the two inverted repeats comprises SEQ ID NO: 6. Insome embodiments, the cargo cassette comprises a promoter selected fromthe group consisting of: CMV, EFS, MND, EF1α, CAGCs, PGK, UBC, U6, H1,and Cumate. In some embodiments, the cargo cassette comprises a CMVpromoter. In some embodiments, the cargo cassette comprises a transgene.In some embodiments, the transgene codes for a protein selected from thegroup consisting of: a cellular receptor, an immunological checkpointprotein, a cytokine, and any combination thereof. In some embodiments,the transgene codes for a cellular receptor selected from the groupconsisting of: a T cell receptor (TCR), a B cell receptor (BCR), achimeric antigen receptor (CAR), or any combination thereof. In someembodiments, the cargo cassette is present in a forward direction. Insome embodiments, the cargo cassette is present in a reverse direction.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent that a term incorporated by reference conflicts with aterm defined herein, this specification controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows the transposition efficiency of several exemplary TcBustertransposon vector constructs, as measured by percent of mCherry positivecells in cells that were transfected with wild-type (WT) TcBustertransposase and the exemplary TcBuster transposons.

FIG. 2 shows nucleotide sequence comparison of exemplary TcBuster IR/DRsequence 1 (SEQ ID NOS 3-4, respectively in order of appearance) andsequence 2 (SEQ ID NOS 5-6, respectively in order of appearance).

FIG. 3A shows representative bright-field and fluorescent images ofHEK-293T cells 2 weeks after transfection with exemplary TcBustertransposon Tn-8 (containing puro-mCherry cassette; illustrated inFIG. 1) and WT TcBuster transposase or V596A mutant transposase(containing V596A substitution). The transfected cells were plated in6-well plate with 1 μg/mL puromycin 2 days posttransfection, and werefixed and stained 2 weeks posttransfection with crystal violet forcolony quantification. FIG. 3B shows representative pictures of thetransfected cell colonies in 6-well plate 2 weeks posttransfection. FIG.3C is a graph showing the quantification of colonies per eachtransfection condition 2 weeks posttransfection.

FIG. 4 depicts the amino acid sequence alignment of TcBuster transposaseversus a number of transposases in AC subfamily, with only regions ofamino acid conservation being shown (SEQ ID NOS 89-194, respectively inorder of appearance).

FIG. 5 depicts the amino acid sequence alignment of TcBuster transposaseversus a number of other transposase members in Buster subfamily (SEQ IDNOS 195-203, respectively in order of appearance). Certain exemplaryamino acid substitutions are indicated above the protein sequences,along with the percentage shown on top of the alignment is thepercentage of other Buster subfamily members that contain the amino acidthat is contemplated being substituted into the TcBuster sequence, andthe percentage shown below is the percentage of other Buster subfamilymembers that contain the canonical TcBuster amino acid at that position.

FIG. 6 shows a vector map of an exemplary expression vector pcDNA-DEST40that was used to test TcBuster transposase mutants.

FIG. 7 is a graph quantifying the transposition efficiency of exemplaryTcBuster transposase mutants, as measured by percent of mCherry positivecells in HEK-293T cells that were transfected with TcBuster transposonTn-8 (illustrated in FIG. 1) with the exemplary transposase mutants.

FIG. 8 depicts one exemplary fusion transposase that contains a DNAsequence specific binding domain and a TcBuster transposase sequencejoined by an optional linker.

FIG. 9 is a graph quantifying the transposition efficiency of exemplaryTcBuster transposases containing different tags as measured by percentof mCherry positive cells in HEK-293T cells that were transfected withTcBuster transposon Tn-8 (illustrated in FIG. 1) with the exemplarytransposases containing the tags.

FIG. 10A is a graph quantifying the transposition efficiency ofexemplary TcBuster transposition systems in human CD3+ T cells asmeasured by percent of GFP positive cells. FIG. 10B is a graphquantifying viability of the transfected T cells 2 and 7 dayspost-transfection by flow cytometry. Data is relative to pulse control.

FIG. 11 shows amino acid sequence of wild-type TcBuster transposase withcertain amino acids annotated (SEQ ID NO: 1).

FIG. 12 shows amino acid sequence of mutant TcBuster transposasecontaining amino acid substitutions D189A/V377T/E469K (SEQ ID NO: 78).

FIG. 13 shows amino acid sequence of mutant TcBuster transposasecontaining amino acid substitutions D189A/V377T/E469K/I452K (SEQ ID NO:79).

FIG. 14 shows amino acid sequence of mutant TcBuster transposasecontaining amino acid substitutions D189A/V377T/E469K/N85S (SEQ ID NO:80).

FIG. 15 shows amino acid sequence of mutant TcBuster transposasecontaining amino acid substitutions D189A/V377T/E469K/A358K (SEQ ID NO:81).

FIG. 16 shows amino acid sequence of mutant TcBuster transposasecontaining amino acid substitutions D189A/V377T/E469K/K573E/E578L (SEQID NO: 13).

DETAILED DESCRIPTION OF THE INVENTION

Overview

DNA transposons can translocate via a non-replicative, ‘cut-and-paste’mechanism. This requires recognition of the two terminal invertedrepeats by a catalytic enzyme, i.e. transposase, which can cleave itstarget and consequently release the DNA transposon from its donortemplate. Upon excision, the DNA transposons may subsequently integrateinto the acceptor DNA that is cleaved by the same transposase. In someof their natural configurations, DNA transposons are flanked by twoinverted repeats and may contain a gene encoding a transposase thatcatalyzes transposition.

For genome editing applications with DNA transposons, it is desirable todesign a transposon to develop a binary system based on two distinctplasmids whereby the transposase is physically separated from thetransposon DNA containing the gene of interest flanked by the invertedrepeats. Co-delivery of the transposon and transposase plasmids into thetarget cells enables transposition via a conventional cut-and-pastemechanism.

TcBuster is a member of the hAT family of DNA transposons. Other membersof the family include Sleeping Beauty and PiggBac. Discussed herein arevarious devices, systems and methods relating to synergistic approachesto enhance gene transfer into human hematopoietic and immune systemcells using hAT family transposon components. The present disclosurerelates to improved hAT transposases, transposon vector sequences,transposase delivery methods, and transposon delivery methods. In oneimplementation, the present study identified specific, universal sitesfor making hyperactive hAT transposases. In another implementation,methods for making minimally sized hAT transposon vector invertedterminal repeats (ITRs) that conserve genomic space are described. Inanother implementation, improved methods to deliver hAT familytransposases as chemically modified in vitro transcribed mRNAs aredescribed. In another implementation, methods to deliver hAT familytransposon vectors as “miniature” circles of DNA are described, in whichvirtually all prokaryotic sequences have been removed by a recombinationmethod. In another implementation, methods to fuse DNA sequence specificbinding domains using transcription activator-like (TAL) domains fusedto the hAT transposases are described. These improvements, individuallyor in combination, can yield unexpectedly high levels of gene transferto the cell types in question and improvements in the delivery oftransposon vectors to sequences of interest.

Mutant TcBuster Transposase

One aspect of the present disclosure provides a mutant TcBustertransposase. A mutant TcBuster transposase may comprise one or moreamino acid substitutions in comparison to a wild-type TcBustertransposase (SEQ ID NO: 1).

A mutant TcBuster transposase can comprise an amino acid sequence havingat least 70% sequence identity to full length sequence of a wild-typeTcBuster transposase (SEQ ID NO: 1). In some embodiments, a mutantTcBuster transposase can comprise an amino acid sequence having at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to full length sequence of a wild-type TcBustertransposase (SEQ ID NO: 1). In some cases, a mutant TcBuster transposasecan comprise an amino acid sequence having at least 98%, at least 98.5%,at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%,at least 99.9%, or at least 99.95% sequence identity to full lengthsequence of a wild-type TcBuster transposase (SEQ ID NO: 1).

A mutant TcBuster transposase can comprise an amino acid sequence havingat least one amino acid different from full length sequence of awild-type TcBuster transposase (SEQ ID NO: 1). In some embodiments, amutant TcBuster transposase can comprise an amino acid sequence havingat least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, or more amino acids different fromfull length sequence of a wild-type TcBuster transposase (SEQ ID NO: 1).In some cases, a mutant TcBuster transposase can comprise an amino acidsequence having at least 5, at least 10, at least 20, at least 30, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 200, or at least 300 amino acid differentfrom full length sequence of a wild-type TcBuster transposase (SEQ IDNO: 1). In some cases, a mutant TcBuster transposase can comprise anamino acid sequence having at most 3, at most 6, at most 12, at most 25,at most 35, at most 45, at most 55, at most 65, at most 75, at most 85,at most 95, at most 150, at most 250, or at most 350 amino aciddifferent from full length sequence of a wild-type TcBuster transposase(SEQ ID NO: 1).

As shown in FIG. 4, typically, a wild-type TcBuster transposase can beregarded as comprising, from N terminus to C terminus, a ZnF-BED domain(amino acids 76-98), a DNA Binding and Oligomerization domain (aminoacids 112-213), a first Catalytic domain (amino acids 213-312), anInsertion domain (amino acids 312-543), and a second Catalytic domain(amino acids 583-620), as well as at least four inter-domain regions inbetween these annotated domains. Unless indicated otherwise, numericalreferences to amino acids, as used herein, are all in accordance to SEQID NO: 1. A mutant TcBuster transposase can comprise one or more aminoacid substitutions in any one of these domains, or any combinationthereof. In some cases, a mutant TcBuster transposase can comprise oneor more amino acid substitutions in ZnF-BED domain, a DNA Binding andOligomerization domain, a first Catalytic domain, an Insertion domain,or a combination thereof. A mutant TcBuster transposase can comprise oneor more amino acid substitutions in at least one of the two catalyticdomains.

An exemplary mutant TcBuster transposase can comprise one or more aminoacid substitutions from Table 1. Sometimes, a mutant TcBustertransposase can comprise at least one of the amino acid substitutionsfrom Table 1. A mutant TcBuster transposase can comprise at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 20, at least 30, or more of the aminoacid substitutions from Table 1.

TABLE 1 Amino Acid of Wild-type TcBuster Transposase (SEQ ID NO: 1)Amino Acid Substitution Q82 Q82E N85 N85S D99 D99A D132 D132A Q151 Q151SQ151 Q151A E153 E153K E153 E153R A154 A154P Y155 Y155H E159 E159A T171T171K T171 T171R K177 K177E D183 D183K D183 D183R D189 D189A T191 T191ES193 S193K S193 S193R Y201 Y201A F202 F202D F202 F202K C203 C203I C203C203V Q221 Q221T M222 M222L I223 I223Q E224 E224G S225 S225W D227 D227AR239 R239H E243 E243A E247 E247K P257 P257K P257 P257R Q258 Q258T E263E263A E263 E263K E263 E263R E274 E274K E274 E274R S278 S278K N281 N281EL282 L282K L282 L282R K292 K292P V297 V297K K299 K299S A303 A303T H322H322E A332 A332S A358 A358E A358 A358K A358 A358S D376 D376A V377 V377TL380 L380N I398 I398D I398 I398S I398 I398K F400 F400L V431 V431L S447S447E N450 N450K N450 N450R I452 I452F E469 E469K P510 P510D P510 P510NE517 E517R R536 R536S V553 V553S P554 P554T P559 P559D P559 P559S P559P559K K573 K573E E578 E578L K590 K590T Y595 Y595L V596 V596A T598 T598IK599 K599A Q615 Q615A T618 T618K T618 T618K T618 T618R D622 D622K D622D622R

An exemplary mutant TcBuster transposase comprises one or more aminoacid substitutions, or combinations of substitutions, from Table 2.Sometimes, a mutant TcBuster transposase can comprise at least one ofthe amino acid substitutions, or combinations of substitutions, fromTable 2. A mutant TcBuster transposase can comprise at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 20, at least 30, or more of the amino acidsubstitutions, or combinations of substitutions, from Table 2.

TABLE 2 Amino Acid of Wild-type TcBuster Transposase (SEQ ID NO: 1)Amino Acid Substitution V377 and E469 V377T/E469K V377, E469, and R536SV377T/E469K/R536S A332 A332S V553 and P554 V553S/P554T E517 E517R K299K299S Q615 and T618 Q615A/T618K S278 S278K A303 A303T P510 P510D P510P510N N281 N281S N281 N281E K590 K590T Q258 Q258T E247 E247K S447 S447EN85 N85S V297 V297K A358 A358K I452 I452F V377, E469, D189V377T/E469K/D189A K573, E578 K573E/E578L I452, V377, E469, D189I452F/V377T/E469K/D189A A358, V377, E469, D189 A358K/V377T/E469K/D189AK573, E578, V377, E469, D189 K573E/E578L/V377T/E469K/D189A T171 T171RD183 D183R S193 S193R P257 P257K E263 E263R L282 L282K T618 T618K D622D622R E153 E153K N450 N450K T171 T171K D183 D183K S193 S193K P257 P257RE263 E263K L282 L282R T618 T618R D622 D622K E153 E153R N450 N450R E247,E274, V297, A358 E247K/E274K/V297K/A358K

An exemplary mutant TcBuster transposase comprises one or more aminoacid substitutions, or combinations of substitutions, from Table 3.Sometimes, a mutant TcBuster transposase can comprise at least one ofthe amino acid substitutions, or combinations of substitutions, fromTable 3. A mutant TcBuster transposase can comprise at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 20, at least 30, or more of the amino acidsubstitutions, or combinations of substitutions, from Table 3.

TABLE 3 Amino Acid of Wild-type TcBuster Transposase (SEQ ID NO: 1)Amino Acid Substitutions V377 and E469 V377T/E469K V377, E469, and R536SV377T/E469K/R536S A332 A332S V553 and P554 V553S/P554T E517 E517R K299K299S Q615 and T618 Q615A/T618K S278 S278K A303 A303T P510 P510D P510P510N N281 N281S N281 N281E K590 K590T Q258 Q258T E247 E247K S447 S447EN85 N85S V297 V297K A358 A358K I452 I452F V377, E469, D189V377T/E469K/D189A K573, E578 K573E/E578L

Hyperactive Mutant TcBuster Transposase

Another aspect of the present disclosure is to provide a hyperactivemutant TcBuster transposase. A “hyperactive” mutant TcBustertransposase, as used herein, can refer to any mutant TcBustertransposase that has increased transposition efficiency as compared to awild-type TcBuster transposase having amino acid sequence SEQ ID NO: 1.

In some embodiments, a hyperactive mutant TcBuster transposase may haveincreased transposition efficiency under certain situations as comparedto a wild-type TcBuster transposase having amino acid sequence SEQ IDNO: 1. For example, the hyperactive mutant TcBuster transposase may havebetter transposition efficiency than the wild-type TcBuster transposasewhen being used to catalyze transposition of transposons havingparticular types of inverted repeat sequences. It is possible that withsome other transposons having other types of inverted repeat sequences,the hyperactive mutant TcBuster transposase does not have increasedtransposition efficiency in comparison to the wild-type TcBustertransposase. In some other non-limiting examples, the hyperactive mutantTcBuster transposase may have increased transposition efficiency incomparison to a wild-type TcBuster transposase having amino acidsequence SEQ ID NO: 1, under certain transfection conditions. Withoutbeing limited, when compared to a wild-type TcBuster transposase, ahyperactive mutant TcBuster transposase may have better transpositionefficiency when the temperature is higher than normal cell culturetemperature; a hyperactive mutant TcBuster transposase may have bettertransposition efficiency in a relative acidic or basic aqueous medium; ahyperactive mutant TcBuster transposase may have better transpositionefficiency when a particular type of transfection technique (e.g.electroporation) is performed.

Transposition efficiency can be measured by the percent of successfultransposition events occurring in a population of host cells normalizedby the amount of transposon and transposase introduced into thepopulation of host cells. In many instances, when the transpositionefficiency of two or more transposases is compared, the same transposonconstruct is paired with each of the two or more transposases fortransfection of the host cells under same or similar transfectionconditions. The amount of transposition events in the host cells can beexamined by various approaches. For example, the transposon constructmay be designed to contain a reporter gene positioned between theinverted repeats, and transfected cells positive for the reporter genecan be counted as the cells where successful transposition eventsoccurs, which can give an estimate of the amount of the transpositionevents. Another non-limiting example includes sequencing of the hostcell genome to examine the insertion of the cassette cargo of thetransposon. In some embodiments, when the transposition efficiency oftwo or more different transposons is compared, the same transposase canbe paired with each of the different transposons for transfection of thehost cells under same or similar transfection conditions. Similarapproaches can be utilized for the measurement of transpositionefficiency. Other methods known to one skilled in the art may also beimplemented for the comparison of transposition efficiency.

Also provided herein are methods of obtaining a hyperactive mutantTcBuster transposase.

One exemplary method can comprise systemically mutating amino acids ofTcBuster transposase to increase a net charge of the amino acidsequence. Sometimes, the method can comprise performing systematicalanine scanning to mutate aspartic acid (D) or glutamic acid (E), whichare negatively charged at a neutral pH, to alanine residues. A methodcan comprise performing systemic mutation to lysing (K) or arginine (R)residues, which are positively charged at a neutral pH.

Without wishing to be bound by a particular theory, increase in a netcharge of the amino acid sequence at a neutral pH may increase thetransposition efficiency of the TcBuster transposase. Particularly, whenthe net charge is increased in proximity to a catalytic domain of thetransposase, the transposition efficiency is expected to increase. Itcan be contemplated that positively charged amino acids can form pointsof contact with DNA target and allow the catalytic domains to act on theDNA target. It may also be contemplated that loss of these positivelycharged amino acids can decrease either excision or integration activityin transposases.

FIG. 11 depicts the WT TcBuster transposase amino acid sequence,highlighting amino acids that may be points of contact with DNA. In FIG.11, large bold lettering indicates catalytic triad amino acids;lettering with boxes indicates amino acids that when substituted to apositive charged amino acid increases transposition; italicized andlowercased lettering indicates positive charged amino acids that whensubstituted to a different amino acid decreases transposition; bolditalicized and underlined indicates amino acids that when substituted toa positive charged amino acid increases transposition, and whensubstituted to a negative charged amino acid decreases transposition;underlined lettering indicates amino acids that could be positivecharged amino acids based on protein sequence alignment to the Bustersubfamily.

A mutant TcBuster transposase can comprise one or more amino acidsubstitutions that increase a net charge at a neutral pH in comparisonto SEQ ID NO: 1. Sometimes, a mutant TcBuster transposase comprising oneor more amino acid substitutions that increase a net charge at a neutralpH in comparison to SEQ ID NO: 1 can be hyperactive. Sometimes, themutant TcBuster transposase can comprise one or more substitutions to apositively charged amino acid, such as, but not limited to, lysine (K)or arginine (R). A mutant TcBuster transposase can comprise one or moresubstitutions of a negatively charged amino acid, such as, but notlimited to, aspartic acid (D) or glutamic acid (E), with a neutral aminoacid, or a positively charged amino acid.

One non-limiting example includes a mutant TcBuster transposase thatcomprises one or more amino acid substitutions that increase a netcharge at a neutral pH within or in proximity to a catalytic domain incomparison to SEQ ID NO: 1. The catalytic domain can be the firstcatalytic domain or the second catalytic domain. The catalytic domaincan also include both catalytic domains of the transposase.

An exemplary method of the present disclosure can comprise mutatingamino acids that are predicted to be in close proximity to, or to makedirect contact with, the DNA. These amino acids can be substituted aminoacids identified as being conserved in other member(s) of the hAT family(e.g., other members of the Buster and/or Ac subfamilies). The aminoacids predicted to be in close proximity to, or to make direct contactwith, the DNA can be identified, for example, by reference to a crystalstructure, predicted structures, mutational analysis, functionalanalysis, alignment with other members of the hAT family, or any othersuitable method.

Without wishing to be bound by a particular theory, TcBustertransposase, like other members of the hAT transposase family, has a DDEmotif, which may be the active site that catalyzes the movement of thetransposon. It is contemplated that D223, D289, and E589 make up theactive site, which is a triad of acidic residues. The DDE motif maycoordinate divalent metal ions and can be important in the catalyticreaction. In some embodiments, a mutant TcBuster transposase cancomprise one or more amino acid substitutions that increase a net chargeat a neutral pH in comparison to SEQ ID NO: 1, and the one or more aminoacids are located in proximity to D223, D289, or E589, when numbered inaccordance to SEQ ID NO: 1.

In certain embodiments, a mutant TcBuster transposase as provided hereindoes not comprise any disruption of the catalytic triad, i.e. D223,D289, or E589. A mutant TcBuster transposase may not comprise any aminoacid substitution at D223, D289, or E589. A mutant TcBuster transposasemay comprises amino acid substitution at D223, D289, or E589, but suchsubstitution does not disrupt the catalytic activity contributed by thecatalytic triad.

In some cases, the term “proximity” can refer to a measurement of alinear distance in the primary structure of the transposase. Forinstance, the distance between D223 and D289 in the primary structure ofa wild-type TcBuster transposase is 66 amino acids. In certainembodiments, the proximity can refer to a distance of about 70 to 80amino acids. In many cases, the proximity can refer to a distance ofabout 80, 75, 70, 60, 50, 40, 30, 20, 10, or 5 amino acids.

In some cases, the term “proximity” can refer to a measurement of aspatial relationship in the secondary or tertiary structure of thetransposase, i.e. when the transposase folds into its three dimensionalconfigurations. Protein secondary structure can refer to threedimensional form of local segments of proteins. Common secondarystructural elements include alpha helices, beta sheets, beta turns andomega loops. Secondary structure elements may form as an intermediatebefore the protein folds into its three dimensional tertiary structure.Protein tertiary structure can refer to the three dimensional shape of aprotein. Protein tertiary structure may exhibit dynamic configurationalchange under physiological or other conditions. The tertiary structurewill have a single polypeptide chain “backbone” with one or more proteinsecondary structures, the protein domains. Amino acid side chains mayinteract and bond in a number of ways. The interactions and bonds ofside chains within a particular protein determine its tertiarystructure. In many implementations, the proximity can refer to adistance of about 1 Å, about 2 Å, about 5 Å, about 8 Å, about 10 Å,about 15 Å, about 20 Å, about 25 Å, about 30 Å, about 35 Å, about 40 Å,about 50 Å, about 60 Å, about 70 Å, about 80 Å, about 90 Å, or about 100Å.

A neutral pH can be a pH value around 7. Sometimes, a neutral pH can bea pH value between 6.9 and 7.1, between 6.8 and 7.2, between 6.7 and7.3, between 6.6 and 7.4, between 6.5 and 7.5, between 6.4 and 7.6,between 6.3 and 7.7, between 6.2-7.8, between 6.1-7.9, between 6.0-8.0,between 5-8, or in a range derived therefrom.

Non-limiting exemplary mutant TcBuster transposases that comprise one ormore amino acid substitutions that increase a net charge at a neutral pHin comparison to SEQ ID NO: 1 include TcBuster transposases comprisingat least one of the combinations of amino acid substitutions from Table4. A mutant TcBuster transposase can comprise at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 20, at least 30, or more of the amino acidsubstitutions from Table 4.

In some embodiments, a mutant TcBuster transposase can comprise one ormore amino acid substitutions that increase a net charge at anon-neutral pH in comparison to SEQ ID NO: 1. In some cases, the netcharge is increased within or in proximity to a catalytic domain at anon-neutral pH. In many cases, the net charge is increased in proximityto D223, D289, or E589, at a non-neutral pH. The non-neutral pH can be apH value lower than 7, lower than 6.5, lower than 6, lower than 5.5,lower than 5, lower than 4.5, lower than 4, lower than 3.5, lower than3, lower than 2.5, lower than 2, lower than 1.5, or lower than 1. Thenon-neutral pH can also be a pH value higher than 7, higher than 7.5,higher than 8, higher than 8.5, higher than 9, higher than 9.5, orhigher than 10.

TABLE 4 Amino Acid of Wild-type TcBuster Transposase (SEQ ID NO: 1)Amino Acid Substitutions E247 E247K E274 E274K V297 V297K A358 A358KS278 S278K E247 E247R E274 E274R V297 V297R A358 A358R S278 S278R T171T171R D183 D183R S193 S193R P257 P257K E263 E263R L282 L282K T618 T618KD622 D622R E153 E153K N450 N450K T171 T171K D183 D183K S193 S193K P257P257R E263 E263K L282 L282R T618 T618R D622 D622K E153 E153R N450 N450R

In one exemplary embodiment, a method can comprise systemically mutatingamino acids in the DNA Binding and Oligomerization domain. Withoutwishing to be bound by a particular theory, mutation in the DNA Bindingand Oligomerization domain may increase the binding affinity to DNAtarget and promote oligomerization activity of the transposase, whichconsequentially may promote transposition efficiency. More specifically,the method can comprise systemically mutating amino acids one by onewithin or in proximity to the DNA Binding and Oligomerization domain(e.g., amino acid 112 to 213). The method can also comprise mutatingmore than one amino acid within or in proximity to the DNA Binding andOligomerization domain. The method can also comprise mutating one ormore amino acids within or in proximity to the DNA Binding andOligomerization domain, together with one or more amino acids outsidethe DNA Binding and Oligomerization domain.

In some embodiments, the method can comprise performing rationalreplacement of selective amino acid residues based on multiple sequencealignments of TcBuster with other hAT family transposases (Ac, Hermes,Hobo, Tag2, Tam3, Hermes, Restless and Tol2) or with other members ofBuster subfamily (e.g., AeBuster1, AeBuster2, AeBuster3, BtBuster1,BtBuster2, CfBuster1, and CfBuster2). Without being bound by a certaintheory, conservancy of certain amino acids among other hAT familytransposases, especially among the active ones, may indicate theirimportance for the catalytic activity of the transposases. Therefore,replacement of unconserved amino acids in wild-type TcBuster sequence(SEQ ID NO: 1) with conserved amino acids among other hAT family mayyield hyperactive mutant TcBuster transposase. The method may compriseobtaining sequences of TcBuster as well as other hAT familytransposases; aligning the sequences and identifying the amino acids inTcBuster transposase with a different conserved counterpart among theother hAT family transposases; performing site-directed mutagenesis toproduce mutant TcBuster transposase harboring the mutation(s).

A hyperactive mutant TcBuster transposase can comprise one or more aminoacid substitutions based on alignment to other members of Bustersubfamily or other members of hAT family. In many cases, the one or moreamino acid substitutions can be substitutions of conserved amino acidfor the unconserved amino acid in wild-type TcBuster sequence (SEQ IDNO: 1). Non-limiting examples of mutant TcBuster transposases includeTcBuster transposases comprising at least one of the amino acidsubstitutions from Table 5. A mutant TcBuster transposase can compriseat least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 20, at least 30, or moreof the amino acid substitutions from Table 5.

Another exemplary method can comprise systemically mutating acidic aminoacids to basic amino acids and identifying hyperactive mutanttransposase.

In some cases, mutant TcBuster transposase can comprise amino acidsubstitutions V377T, E469K, and D189A. A mutant TcBuster transposase cancomprise amino acid substitutions K573E and E578L. A mutant TcBustertransposase can comprise amino acid substitution I452K. A mutantTcBuster transposase can comprise amino acid substitution A358K. Amutant TcBuster transposase can comprise amino acid substitution V297K.A mutant TcBuster transposase can comprise amino acid substitution N85S.A mutant TcBuster transposase can comprise amino acid substitutionsN85S, V377T, E469K, and D189A. A mutant TcBuster transposase cancomprise amino acid substitutions I452F, V377T, E469K, and D189A. Amutant TcBuster transposase can comprise amino acid substitutions A358K,V377T, E469K, and D189A. A mutant TcBuster transposase can compriseamino acid substitutions V377T, E469K, D189A, K573E and E578L.

TABLE 5 Amino Acid of Wild-type TcBuster Transposase (SEQ ID NO: 1)Amino Acid Substitution Q151 Q151S Q151 Q151A A154 A154P Q615 Q615A V553V553S Y155 Y155H Y201 Y201A F202 F202D F202 F202K C203 C203I C203 C203VF400 F400L I398 I398D I398 I398S I398 I398K V431 V431L P559 P559D P559P559S P559 P559K M222 M222L

Fusion Transposase

Another aspect of the present invention provides a fusion transposase.The fusion transposase can comprise a TcBuster transposase sequence anda DNA sequence specific binding domain.

The TcBuster transposase sequence of a fusion transposase can comprisean amino acid sequence of any of the mutant TcBuster transposases asdescribed herein. The TcBuster transposase sequence of a fusiontransposase can also comprise an amino acid sequence of a wild-typeTcBuster transposase having amino acid sequence SEQ ID NO: 1.

A DNA sequence specific binding domain as described herein can refer toa protein domain that is adapted to bind to a DNA molecule at a sequenceregion (“target sequence”) containing a specific sequence motif. Forinstance, an exemplary DNA sequence specific binding domain mayselectively bind to a sequence motif TATA, while another exemplary DNAsequence specific binding domain may selectively bind to a differentsequence motif ATGCNTAGAT (SEQ ID NO: 82) (N denotes any one of A, T, G,and C).

A fusion transposase as provided herein may direct sequence specificinsertion of the transposon. For instance, a DNA sequence specificbinding domain may guide the fusion transposase to bind to a targetsequence based on the binding specificity of the binding domain. Beingbound to or restricted to a certain sequence region may spatially limitthe interaction between the fusion transposase and the transposon,thereby limiting the catalyzed transposition to a sequence region inproximity to the target sequence. Depending on the size,three-dimensional configuration, and sequence binding affinity of theDNA binding domain, as well as the spatial relationship between the DNAbinding domain and the TcBuster transposase sequence, and theflexibility of the connection between the two domains, the distance ofthe actual transposition site to the target sequence may vary. Properdesign of the fusion transposase configuration can direct thetransposition to a desirable target genomic region.

A target genomic region for transposition can be any particular genomicregion, depending on application purposes. For instance, sometimes, itis desirable to avoid transcription start sites for the transposition,which may cause undesirable, or even harmful, change in expression levelof certain important endogenous gene(s) of the cell. A fusiontransposase may contain a DNA sequence specific binding domain that cantarget the transposition to a safe harbor of the host genome.Non-limiting examples of safe harbors can include HPRT, AAVS site (e.g.AAVS1, AAVS2, ETC.), CCR5, or Rosa26. Safe harbor sites can generallyrefer to sites for transgene insertion whose use exert little to nonedisrupting effects on genome integrity of the cell or cellular healthand functions.

A DNA sequence specific binding domain may be derived from, or be avariant of any DNA binding protein that has sequence-specificity. Inmany instances, a DNA sequence specific binding domain may comprise anamino acid sequence at least 40%, at least 50%, at least 60%, atleast70%, at least 80%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100% identical to a naturally occurring sequence-specificDNA binding protein. A DNA sequence specific binding domain may comprisean amino acid sequence at least 70% identical to a naturally occurringsequence-specific DNA binding protein. Non-limiting examples of anaturally occurring sequence-specific DNA binding protein include, butnot limited to, transcription factors from various origins,specific-sequence nucleases, and viral replication proteins. A naturallyoccurring sequence-specific DNA binding protein can also be any otherprotein having the specific binding capability from various origins.Selection and prediction of DNA binding proteins can be conducted byvarious approaches, including, but not limited to, using computationalprediction databases available online, like DP-Bindlcg<dot>rit<dot>Albany<dot>edu</>dp-bind) or DNABINDdnabind<dot>szialab<dot>org).

The term “transcription factor” can refer to a protein that controls therate of transcription of genetic information from DNA to messenger DNA,by binding to a specific DNA sequence. A transcription factor that canbe used in a fusion transposase described herein can be based on aprokaryotic transcription factor or a eukaryotic transcription factor,as long as it confers sequence specificity when binding to the targetDNA molecule. Transcription factor prediction databases such as DBD(transcriptionfactor<dot>org) may be used for selection of appropriatetranscription factor for application of the disclosure herein.

A DNA sequence specific binding domain as used herein can comprise oneor more DNA binding domain from a naturally occurring transcriptionfactor. Non-limiting examples of DNA binding domains of transcriptionfactors include DNA binding domains that belong to families like basichelix-loop-helix, basic-leucine zipper (bZIP), C-terminal effectordomain of the bipartite response regulators, AP2/ERF/GCC box,helix-turn-helix, homeodomain proteins, lambda repressor-like, srf-like(serum response factor), paired box, winged helix, zinc fingers,multi-domain Cys2His2 (C2H2) zinc fingers, Zn2/Cys6, or Zn2/Cys8 nuclearreceptor zinc finger.

A DNA sequence specific binding domain can be an artificially engineeredamino acid sequence that binds to specific DNA sequences. Non-limitingexamples of such artificially designed amino acid sequence includesequences created based on frameworks like transcription activator likeeffector nucleases (TALEs) DNA binding domain, zinc finger nucleases,adeno associated virus (AAV) Rep protein, and any other suitable DNAbinding proteins as described herein.

Natural TALEs are proteins secreted by Xanthomonas bacteria to aid theinfection of plant species. Natural TALEs can assist infections bybinding to specific DNA sequences and activating the expression of hostgenes. In general, TALE proteins consist of a central repeat domain,which determines the DNA targeting specificity and can be rapidlysynthesized de novo. TALEs have a modular DNA-binding domain (DBD)containing repetitive sequences of residues. In some TALEs, each repeatregion contains 34 amino acids. The term “TALE domain” as used hereincan refer to the modular DBD of TALEs. A pair of residues at the 12thand 13th position of each repeat region can determine the nucleotidespecificity and are referred to as the repeat variable diresidue (RVD).The last repeat region, termed the half-repeat, is typically truncatedto 20 amino acids. Combining these repeat regions allows synthesizingsequence-specific synthetic TALEs. The C-terminus typically contains anuclear localization signal (NLS), which directs a TALE to the nucleus,as well as a functional domain that modulates transcription, such as anacidic activation domain (AD). The endogenous NLS can be replaced by anorganism-specific localization signal. For example, an NLS derived fromthe simian virus 40 large T-antigen can be used in mammalian cells. TheRVDs HD, NG, NI, and NN target C, T, A, and G/A, respectively. A list ofRVDs and their binding preferences under certain circumstances fornucleotides can be found in Table 6. Additional TALE RVDs can also beused for custom degenerate TALE-DNA interactions. For example, NA hashigh affinity for all four bases of DNA. Additionally, N*, where * is anRVD with a deletion in the 13th residue, can accommodate all letters ofDNA including methylated cytosine. Also S* may have the ability to bindto any DNA nucleotide.

A number of online tools are available for designing TALEs to target aspecific DNA sequence, for example TALE-NT(tale-nt<dot>cac<dot>cornell<dot>edu), Mojo hand (talendesign<dot>org).Commercially available kits may also assist in creating custom assemblyof TALE repeat regions between the N and C-terminus of the protein.These methods can be used to assemble custom DBDs, which are then clonedinto an expression vector containing a functional domain, e.g. TcBustertransposase sequence.

TABLE 6 RVD Binding Preference nucleotides RVD A G C T NN medium mediumNK weak NI medium NG weak HD medium NS weak medium weak weak NG weak N*weak weak HN weak medium NT weak medium NP weak weak medium NH medium SNweak SH weak NA weak strong weak weak IG weak H* poor poor weak poor NDweak HI medium HG weak NC weak NQ weak SS weak SN weak S* medium mediumstrong medium NV weak medium poor poor HH poor poor poor poor YG poorpoor poor poor

TALEs can be synthesized de novo in the laboratory, for example, bycombining digestion and ligation steps in a Golden Gate reaction withtype II restriction enzymes. Alternatively, TALE can be assembled by anumber of different approaches, including, but not limited to,Ligation-Independent Cloning (LIC), Fast Ligation-based AutomatableSolid-phase High-throughput (FLASH) assembly, and Iterative-CappedAssembly (ICA).

Zinc fingers (ZF) are ˜30 amino acids that can bind to a limitedcombination of ˜3 nucleotides. The C2H2 ZF domain may be the most commontype of ZF and appears to be one of the most abundantly expressedproteins in eukaryotic cells. ZFs are small, functional andindependently folded domains coordinated with zinc molecules in theirstructure. Amino acids in each ZF can have affinity towards specificnucleotides, causing each finger to selectively recognize 3-4nucleotides of DNA. Multiple ZFs can be arranged into a tandem array andrecognize a set of nucleotides on the DNA. By using a combination ofdifferent zinc fingers, a unique DNA sequence within the genome can betargeted. Different ZFPs of various lengths can be generated, which mayallow for recognition of almost any desired DNA sequence out of thepossible 64 triplet subsites.

Zinc fingers to be used in connection with the present disclosure can becreated using established modular assembly fingers, such as a set ofmodular assembly finger domains developed by Barbas and colleagues, andalso another set of modular assembly finger domains by ToolGen. Both setof domains cover all 3 bp GNN, most ANN, many CNN and some TNN triplets(where N can be any of the four nucleotides). Both have a different setof fingers, which allows for searching and coding different ZF modulesas needed. A combinatorial selection-based oligomerized pool engineering(OPEN) strategy can also be employed to minimize context-dependenteffects of modular assembly involving the position of a finger in theprotein and the sequence of neighboring fingers. OPEN ZF arrays arepublicly available from the Zinc Finger Consortium Database.

AAV Rep DNA-binding domain is another DNA sequence specific bindingdomain that can be used in connection with the subject matter of thepresent disclosure. Viral cis-acting inverted terminal repeats (ITRs),and the trans-acting viral Rep proteins (Rep) are believed to be thefactors mediating preferential integration of AAV into AAVS1 site of thehost genome in the absence of a helper virus. AAV Rep protein can bindto specific DNA sequence in the AAVS1 site. Therefore, a site-specificDNA-binding domain can be fused together with a TcBuster transposasedomain as described herein.

A fusion transposase as provided herein can comprise a TcBustertransposase sequence and a tag sequence. A tag sequence as provideherein can refer to any protein sequence that can be used as a detectiontag of the fusion protein, such as, but not limited to, reporterproteins and affinity tags that can be recognized by antibodies.Reporter proteins include, but not limited to, fluorescent proteins(e.g. GFP, RFP, mCherry, YFP), β-galactosidase (β-gal), alkalinephosphatase (AP), chloramphenicol acetyl transferase (CAT), horseradishperoxidase (HRP). Non-limiting examples of affinity tags includepolyhistidine (His tag), Glutathione S-Transferase (GST), MaltoseBinding Protein (MBP), Calmodulin Binding Peptide (CBP), intein-chitinbinding domain (intein-CBD), Streptavidin/Biotin-based tags, Epitopetags like FLAG, HA, c-myc, T7, Glu-Glu and many others.

A fusion transposase as provided herein can comprise a TcBustertransposase sequence and a DNA sequence specific binding domain or a tagsequence fused together without any intermediate sequence (e.g.,“back-to-back”). In some cases, a fusion transposase as provided hereincan comprise a TcBuster transposase sequence and a DNA sequence specificbinding domain or a tag sequence joined by a linker sequence. FIG. 8 isa schematic of an exemplary fusion transposase that comprises a DNAsequence specific binding domain and a TcBuster transposase sequence,joined by a linker. In an exemplary fusion transposase, a linker mayserve primarily as a spacer between the first and second polypeptides. Alinker can be a short amino acid sequence to separate multiple domainsin a single polypeptide. A linker sequence can comprise linkersoccurring in natural multi-domain proteins. In some instances, a linkersequence can comprise linkers artificially created. The choice of linkersequence may be based on the application of the fusion transposase. Alinker sequence can comprise 3, 4, 5, 6, 7, 8, 9, 10, or more aminoacids. In some embodiments, the linker sequence may comprise at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 15, at least 20, or at least 50 amino acids. Insome embodiments, the linker sequence can comprise at most 4, at most 5,at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, atmost 12, at most 15, at most 20, at most 30, at most 40, at most 50, orat most 100 amino acids. In certain cases, it may be desirable to useflexible linker sequences, such as, but not limited to, stretches of Glyand Ser residues (“GS” linker) like (GGGGS)n (n=2-8) (SEQ ID NO: 83),(Gly)₈ (SEQ ID NO: 84), GSAGSAAGSGEF (SEQ ID NO: 85), (GGGGS)₄ (SEQ IDNO: 86). Sometimes, it may be desirable to use rigid linker sequences,such as, but not limited to, (EAAAK)n (n=2-7) (SEQ ID NO: 87), Pro-richsequences like (XP)n, with X designating any amino acid.

In an exemplary fusion transposase provided herein, a TcBustertransposase sequence can be fused to the N-terminus of a DNA sequencespecific binding domain or a tag sequence. Alternatively, a TcBustertransposase sequence can be fused to the C-terminus of a DNA sequencespecific binding domain or a tag sequence. In some embodiments, a thirddomain sequence or more of other sequences can be present in between theTcBuster transposase and the DNA sequence specific binding domain or thetag sequence, depending on the application of the fusion transposase.

TcBuster Transposon

Another aspect of the present disclosure provides a TcBuster transposonthat comprises a cassette cargo positioned between two inverted repeats.A TcBuster transposon can be recognized by a TcBuster transposase asdescribed herein, e.g., a TcBuster transposase can recognize theTcBuster transposon and catalyze transposition of the TcBustertransposon into a DNA sequence.

The terms “inverted repeats”, “terminal inverted repeats”, “invertedterminal repeats”, as used interchangeably herein, can refer to shortsequence repeats flanking the transposase gene in a natural transposonor a cassette cargo in an artificially engineered transposon. The twoinverted repeats are generally required for the mobilization of thetransposon in the presence of a corresponding transposase. Invertedrepeats as described herein may contain one or more direct repeat (DR)sequences. These sequences usually are embedded in the terminal invertedrepeats (TIRs) of the elements. The term “cargo cassette” as used hereincan refer to a nucleotide sequence other than a native nucleotidesequence between the inverted repeats that contains the TcBustertransposase gene. A cargo cassette can be artificially engineered.

A transposon described herein may contain a cargo cassette flanked byIR/DR sequences. In some embodiments, at least one of the repeatscontains at least one direct repeat. As shown in FIGS. 1 and 2, atransposon may contain a cargo cassette flanked by IRDR-L-Seq1 (SEQ IDNO: 3) and IRDR-R-Seq1 (SEQ ID NO: 4). In many cases, a left invertedrepeat can comprise a sequence at least 40%, at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100% identical to IRDR-L-Seq1 (SEQ ID NO: 3). Sometimes, aright inverted repeat can comprise a sequence at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, at least 99%, or 100% identical to IRDR-R-Seq1 (SEQID NO: 4). In other cases, a right inverted repeat can comprise asequence at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, at least 99%, or100% identical to IRDR-L-Seq1 (SEQ ID NO: 3). Sometimes, a left invertedrepeat can comprise a sequence at least 40%, at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100% identical to IRDR-R-Seq1 (SEQ ID NO: 4). The terms“left” and “right”, as used herein, can refer to the 5′ and 3′ sides ofthe cargo cassette on the sense strand of the double strand transposon,respectively. It is also possible that a transposon may contain a cargocassette flanked by IRDR-L-Seq2 (SEQ ID NO: 5) and IRDR-R-Seq2 (SEQ IDNO: 6). In many cases, a left inverted repeat can comprise a sequence atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% identicalto IRDR-L-Seq2 (SEQ ID NO: 5). Sometimes, a right inverted repeat cancomprise a sequence at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 98%, at least99%, or 100% identical to IRDR-R-Seq2 (SEQ ID NO: 6). In other cases, aright inverted repeat can comprise a sequence at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, at least 99%, or 100% identical to IRDR-L-Seq2 (SEQID NO: 5). Sometimes a left inverted repeat can comprise a sequence atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% identicalto IRDR-R-Seq2 (SEQ ID NO: 6). A transposon may contain a cargo cassetteflanked by two inverted repeats that have different nucleotide sequencesthan the ones given in FIG. 2, or a combination of the various sequencesknown to one skilled in the art. At least one of the two invertedrepeats of a transposon described herein may contain a sequence that isat least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% identicalto any one of SEQ ID NOs: 3-6. At least one of inverted repeats of atransposon described herein may contain a sequence that is at least 80%identical to SEQ ID NO: 3 or 4. At least one of inverted repeats of atransposon described herein may contain a sequence that is at least 80%identical to SEQ ID NO: 5 or 6. The choice of inverted repeat sequencesmay vary depending on the expected transposition efficiency, the type ofcell to be modified, the transposase to use, and many other factors.

In many implementations, minimally sized transposon vector invertedterminal repeats that conserve genomic space may be used. The ITRs ofhAT family transposons diverge greatly with differences in right-handand left-hand ITRs. In many cases, smaller ITRs consisting of just100-200 nucleotides are as active as the longer native ITRs in hATtransposon vectors. These sequences may be consistently reduced whilemediating hAT family transposition. These shorter ITRs can conservegenomic space within hAT transposon vectors.

The inverted repeats of a transposon provided herein can be about 50 to2000 nucleotides, about 50 to 1000 nucleotides, about 50 to 800nucleotides, about 50 to 600 nucleotides, about 50 to 500 nucleotides,about 50 to 400 nucleotides, about 50 to 350 nucleotides, about 50 to300 nucleotides, about 50 to 250 nucleotides, about 50 to 200nucleotides, about 50 to 180 nucleotides, about 50 to 160 nucleotides,about 50 to 140 nucleotides, about 50 to 120 nucleotides, about 50 to110 nucleotides, about 50 to 100 nucleotides, about 50 to 90nucleotides, about 50 to 80 nucleotides, about 50 to 70 nucleotides,about 50 to 60 nucleotides, about 75 to 750 nucleotides, about 75 to 450nucleotides, about 75 to 325 nucleotides, about 75 to 250 nucleotides,about 75 to 150 nucleotides, about 75 to 95 nucleotides, about 100 to500 nucleotides, about 100 to 400 nucleotides, about 100 to 350nucleotides, about 100 to 300 nucleotides, about 100 to 250 nucleotides,about 100 to 220 nucleotides, about 100 to 200 nucleotides, or in anyrange derived therefrom.

In some cases, a cargo cassette can comprise a promoter, a transgene, ora combination thereof. In cargo cassettes comprising both a promoter anda transgene, the expression of the transgene can be directed by thepromoter. A promoter can be any type of promoter available to oneskilled in the art. Non-limiting examples of the promoters that can beused in a TcBuster transposon include EFS, CMV, MND, EF1α, CAGGs, PGK,UBC, U6, H1, and Cumate. The choice of a promoter to be used in aTcBuster transposition would depend on a number of factors, such as, butnot limited to, the expression efficiency of the promoter, the type ofcell to be genetically modified, and the desired transgene expressionlevel.

A transgene in a TcBuster transposon can be any gene of interest andavailable to one skilled in the art. A transgene can be derived from, ora variant of, a gene in nature, or can be artificially designed. Atransgene can be of the same species origin as the cell to be modified,or from different species. A transgene can be a prokaryotic gene, or aeukaryotic gene. Sometimes, a transgene can be a gene derived from anon-human animal, a plant, or a human being. A transgene can compriseintrons. Alternatively, a transgene may have introns removed or notpresent.

In some embodiments, a transgene can code for a protein. Exemplaryproteins include, but are not limited to, a cellular receptor, animmunological checkpoint protein, a cytokine, or any combinationthereof. Sometimes, a cellular receptor as described herein can include,but not limited to a T cell receptor (TCR), a B cell receptor (BCR), achimeric antigen receptor (CAR), or any combination thereof.

A cargo cassette as described herein may not contain a transgene codingfor any type of protein product, but that is useful for other purposes.For instance, a cargo cassette may be used for creating frameshift inthe insertion site, for example, when it is inserted in an exon of agene in the host genome. This may lead to a truncation of the geneproduct or a null mutation. Sometimes, a cargo cassette may be used forreplacing an endogenous genomic sequence with an exogenous nucleotidesequence, thereby modifying the host genome.

A transposon described herein may have a cargo cassette in eitherforward or reverse direction. In many cases, a cargo cassette has itsown directionality. For instance, a cargo cassette containing atransgene would have a 5′ to 3′ coding sequence. A cargo cassettecontaining a promoter and a gene insertion would have promoter on the 5′site of the gene insertion. The term “forward direction”, as usedherein, can refer to the situation where a cargo cassette maintains itsdirectionality on the sense strand of the double strand transposon. Theterm “reverse direction”, as used herein, can refer to the situationwhere a cargo cassette maintains its directionality on the antisensestrand of the double strand transposon.

Systems for Genome Editing and Methods of Use

Another aspect of the present disclosure provides a system for genomeediting. A system can comprise a TcBuster transposase and a TcBustertransposon. A system can be used to edit a genome of a host cell,disrupting or modifying an endogenous genomic region of the host cell,inserting an exogenous gene into the host genome, replacing anendogenous nucleotide sequence with an exogenous nucleotide sequence orany combination thereof.

A system for genome editing can comprise a mutant TcBuster transposaseor fusion transposase as described herein, and a transposon recognizableby the mutant TcBuster transposase or the fusion transposase. A mutantTcBuster transposase or the fusion transposase can be provided as apurified protein. Protein production and purification technologies areknown to one skilled in the art. The purified protein can be kept in adifferent container than the transposon, or they can be kept in the samecontainer.

In many cases, a system for genome editing can comprise a polynucleotideencoding a mutant TcBuster transposase or fusion transposase asdescribed herein, and a transposon recognizable by the mutant TcBustertransposase or the fusion transposase. Sometimes, a polynucleotide ofthe system can comprise DNA that encodes the mutant TcBuster transposaseor the fusion transposase. Alternatively or additionally, apolynucleotide of the system can comprise messenger RNA (mRNA) thatencodes the mutant TcBuster transposase or the fusion transposase. ThemRNA can be produced by a number of approaches well known to one ofordinary skills in the art, such as, but not limited to, in vivotranscription and RNA purification, in vitro transcription, and de novosynthesis. In many cases, the mRNA can be chemically modified. Thechemically modified mRNA may be resistant to degradation than unmodifiedor natural mRNAs or may degrade more quickly. In many cases, thechemical modification of the mRNA may render the mRNA being translatedwith more efficiency. Chemical modification of mRNAs can be performedwith well-known technologies available to one skilled in the art, or bycommercial vendors.

For many applications, safety dictates that the duration of hATtransposase expression be only long enough to mediate safe transposondelivery. Moreover, a pulse of hAT transposase expression that coincideswith the height of transposon vector levels can achieve maximal genedelivery. The implementations are made using available technologies forthe in vitro transcription of RNA molecules from DNA plasmid templates.The RNA molecules can be synthesized using a variety of methods for invitro (e.g., cell free) transcription from a DNA copy. Methods to dothis have been described and are commercially available. For example,the mMessage Machine in vitro transcription kit available through lifetechnologies.

There are also a number of companies that can perform in vitrotranscription on a fee for service basis. We have also found that thatchemically modified RNAs for hAT expression work especially well forgene transfer. These chemically modified RNAs do not induce cellularimmune responses and RNA generated using proprietary methods that alsoavoid the cellular immune response. These RNA preparations remove RNAdimers (Clean-Cap) and cellular reactivity (pseudouridine incorporation)produce better transient gene expression in human T cells withouttoxicity in our hands (data not shown). The RNA molecules can beintroduced into cells using any of many described methods for RNAtransfection, which is usually non-toxic to most cells. Methods to dothis have been described and are commercially available. For example,the Amaxa nucleofector, Neon electroporator, and the Maxcyte platforms.

A transposon as described herein may be present in an expression vector.In many cases, the expression vector can be DNA plasmid. Sometimes, theexpression vector can be a mini-circle vector. The term “mini-circlevector” as used herein can refer to small circular plasmid derivativethat is free of most, if not all, prokaryotic vector parts (e.g.,control sequences or non-functional sequences of prokaryotic origin).Under circumstances, the toxicity to the cells created by transfectionor electroporation can be mitigated by using the “mini-circles” asdescribed herein.

A mini-circle vector can be prepared by well-known molecular cloningtechnologies available. First, a ‘parental plasmid’ (bacterial plasmidwith insertion, such as transposon construct) in bacterial, such as E.coli, can be produced, which can be followed by induction of asite-specific recombinase. These steps can then be followed by theexcision of prokaryotic vector parts via two recombinase-targetsequences at both ends of the insert, as well as recovery of theresulting mini-circle vector. The purified mini-circle can betransferred into the recipient cell by transfection or lipofection andinto a differentiated tissue by, for instance, jet injection. Amini-circle containing TcBuster transposon can have a size about 1.5 kb,about 2 kb, about 2.2 kb, about 2.4 kb, about 2.6 kb, about 2.8 kb,about 3 kb, about 3.2 kb, about 3.4 kb, about 3.6 kb, about 3.8 kb,about 4 kb, about 4.2 kb, about 4.4 kb, about 4.6 kb, about 4.8 kb,about 5 kb, about 5.2 kb, about 5.4 kb, about 5.6 kb, about 5.8 kb,about 6 kb, about 6.5 kb, about 7 kb, about 8 kb, about 9 kb, about 10kb, about 12 kb, about 25 kb, about 50 kb, or a value between any two ofthese numbers. Sometimes, a mini-circle containing TcBuster transposonas provided herein can have a size at most 2.1 kb, at most 3.1 kb, atmost 4.1 kb, at most 4.5 kb, at most 5.1 kb, at most 5.5 kb, at most 6.5kb, at most 7.5 kb, at most 8.5 kb, at most 9.5 kb, at most 11 kb, atmost 13 kb, at most 15 kb, at most 30 kb, or at most 60 kb.

In certain embodiments, a system as described herein may contain apolynucleotide encoding a mutant TcBuster transposase or fusiontransposase as described herein, and a transposon, which are present ina same expression vector, e.g. plasmid.

Yet another aspect of the present disclosure provides a method ofgenetic engineering. A method of genetic engineering can compriseintroducing into a cell a TcBuster transposase and a transposonrecognizable by the TcBuster transposase. A method of geneticengineering can also be performed in a cell-free environment. A methodof genetic engineering in a cell-free environment can comprise combininga TcBuster transposase, a transposon recognizable by the transposase,and a target nucleic acid into a container, such as a well or tube.

A method described herein can comprises introducing into a cell a mutantTcBuster transposase provided herein and a transposon recognizable bythe mutant TcBuster transposase. A method of genome editing cancomprise: introducing into a cell a fusion transposase provided hereinand a transposon recognizable by the fusion transposase.

The mutant TcBuster transposase or the fusion transposase can beintroduced into the cell either as a protein or via a polynucleotidethat encodes for the mutant TcBuster transposase or the fusiontransposase. The polynucleotide, as discussed above, can comprise a DNAor an mRNA that encodes the mutant TcBuster transposase or the fusiontransposase.

In many instances, the TcBuster transposase or the fusion transposasecan be transfected into a host cell as a protein, and the concentrationof the protein can be at least 0.05 nM, at least 0.1 nM, at least 0.2nM, at least 0.5 nM, at least 1 nM, at least 2 nM, at least 5 nM, atleast 10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least500 nM, at least 1 μM, at least 2 μM, at least 5 μM, at least 7.5 μM, atleast 10 μM, at least 15 μM, at least 20 μM, at least 25 μM, at least 50μM, at least 100 μM, at least 200 μM, at least 500 μM, or at least 1 μM.Sometimes, the concentration of the protein can be around 1 μM to around50 μM, around 2 μM to around 25 μM, around 5 μM to around 12.5 μM, oraround 7.5 μM to around 10 μM.

In many cases, the TcBuster transposase or the fusion transposase can betransfected into a host cell through a polynucleotide, and theconcentration of the polynucleotide can be at least about 5 ng/ml, 10ng/ml, 20 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 80 ng/ml, 100 ng/ml, 120ng/ml, 150 ng/ml, 180 ng/ml, 200 ng/ml, 220 ng/ml, 250 ng/ml, 280 ng/ml,300 ng/ml, 500 ng/ml, 750 ng/ml, 1 μg/ml, 2 μg/ml, 3 μg/ml, 5 μg/ml, 50μg/ml, 100 μg/ml, 150 μg/ml, 200 μg/ml, 250 μg/ml, 300 μg/ml, 350 μg/ml,400 μg/ml, 450 μg/ml, 500 μg/ml, 550 μg/ml, 600 μg/ml, 650 μg/ml, 700μg/ml, 750 μg/ml, or 800 μg/ml. Sometimes, the concentration of thepolynucleotide can be between about 5-25 μg/ml, 25-50 μg/ml, 50-100μg/ml, 100-150 μg/ml, 150-200 μg/ml, 200-250 μg/ml, 250-500 μg/ml, 5-800μg/ml, 200-800 μg/ml, 250-800 μg/ml, 400-800 μg/ml, 500-800 μg/ml, orany range derivable therein. In many cases, the transposon is present ina separate expression vector than the transposase, and the concentrationof the transposon can be at least about 5 ng/ml, 10 ng/ml, 20 ng/ml, 40ng/ml, 50 ng/ml, 60 ng/ml, 80 ng/ml, 100 ng/ml, 120 ng/ml, 150 ng/ml,180 ng/ml, 200 ng/ml, 220 ng/ml, 250 ng/ml, 280 ng/ml, 300 ng/ml, 500ng/ml, 750 ng/ml, 1 μg/ml, 2 μg/ml, 3 μg/ml, 5 μg/ml, 50 μg/ml, 100μg/ml, 150 μg/ml, 200 μg/ml, 250 μg/ml, 300 μg/ml, 350 μg/ml, 400 μg/ml,450 μg/ml, 500 μg/ml, 550 μg/ml, 600 μg/ml, 650 μg/ml, 700 μg/ml, 750μg/ml, or 800 μg/ml. Sometimes, the concentration of the transposon canbe between about 5-25 μg/ml, 25-50 μg/ml, 50-100 μg/ml, 100-150 μg/ml,150-200 μg/ml, 200-250 μg/ml, 250-500 μg/ml, 5-800 μg/ml, 200-800 μg/ml,250-800 μg/ml, 400-800 μg/ml, 500-800 μg/ml, or any range derivabletherein. It is possible the ratio of the transposon versus thepolynucleotide coding for the transposase is at most 10000, at most5000, at most 1000, at most 500, at most 200, at most 100, at most 50,at most 20, at most 10, at most 5, at most 2, at most 1, at most 0.1, atmost 0.05, at most 0.01, at most 0.001, at most 0.0001, or any number inbetween any two thereof.

In some other cases, the transposon and the polynucleotide coding forthe transposase are present in the same expression vector, and theconcentration of the expression vector containing both transposon andthe polynucleotide encoding transposase can be at least about 5 ng/ml,10 ng/ml, 20 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 80 ng/ml, 100 ng/ml,120 ng/ml, 150 ng/ml, 180 ng/ml, 200 ng/ml, 220 ng/ml, 250 ng/ml, 280ng/ml, 300 ng/ml, 500 ng/ml, 750 ng/ml, 1 μg/ml, 2 μg/ml, 3 μg/ml, 5μg/ml, 50 μg/ml, 100 μg/ml, 150 μg/ml, 200 μg/ml, 250 μg/ml, 300 μg/ml,350 μg/ml, 400 μg/ml, 450 μg/ml, 500 μg/ml, 550 μg/ml, 600 μg/ml, 650μg/ml, 700 μg/ml, 750 μg/ml, or 800 μg/ml. Sometimes, the concentrationof the expression vector containing both transposon and thepolynucleotide encoding transposase can be between about 5-25 μg/ml,25-50 μg/ml, 50-100 μg/ml, 100-150 μg/ml, 150-200 μg/ml, 200-250 μg/ml,250-500 μg/ml, 5-800 μg/ml, 200-800 μg/ml, 250-800 μg/ml, 400-800 μg/ml,500-800 μg/ml, or any range derivable therein.

In some cases, the amount of polynucleic acids that may be introducedinto the cell by electroporation may be varied to optimize transfectionefficiency and/or cell viability. In some cases, less than about 100 pgof nucleic acid may be added to each cell sample (e.g., one or morecells being electroporated). In some cases, at least about 100 pg, atleast about 200 pg, at least about 300 pg, at least about 400 pg, atleast about 500 pg, at least about 600 pg, at least about 700 pg, atleast about 800 pg, at least about 900 pg, at least about 1 microgram,at least about 1.5 μg, at least about 2 μg, at least about 2.5 μg, atleast about 3 μg, at least about 3.5 μg, at least about 4 μg, at leastabout 4.5 μg, at least about 5 μg, at least about 5.5 μg, at least about6 μg, at least about 6.5 μg, at least about 7 μg, at least about 7.5 μg,at least about 8 μg, at least about 8.5 μg, at least about 9 μg, atleast about 9.5 μg, at least about 10 μg, at least about 11 μg, at leastabout 12 μg, at least about 13 μg, at least about 14 μg, at least about15 μg, at least about 20 μg, at least about 25 μg, at least about 30 μg,at least about 35 μg, at least about 40 μg, at least about 45 μg, or atleast about 50 μg, of nucleic acid may be added to each cell sample(e.g., one or more cells being electroporated). For example, 1 microgramof dsDNA may be added to each cell sample for electroporation. In somecases, the amount of polynucleic acids (e.g., dsDNA) required foroptimal transfection efficiency and/or cell viability may be specific tothe cell type.

The subject matter disclosed herein may find use in genome editing of awide range of various types of host cells. In preferred embodiments, thehost cells may be from eukaryotic organisms. In some embodiments, thecells may be from a mammal origin. In some embodiments, the cells may befrom a human origin.

In general, the cells may be from an immortalized cell line or primarycells.

The terms “cell line” and “immortalized cell line”, as used hereininterchangeably, can refer to a population of cells from an organismwhich would normally not proliferate indefinitely but, due to mutation,may have evaded normal cellular senescence and instead can keepundergoing division. The subject matter provided herein may find use ina range of common established cell lines, including, but not limited to,human BC-1 cells, human BJAB cells, human IM-9 cells, human Jiyoyecells, human K-562 cells, human LCL cells, mouse MPC-11 cells, humanRaji cells, human Ramos cells, mouse Ramos cells, human RPMI8226 cells,human RS4-11 cells, human SKW6.4 cells, human Dendritic cells, mouseP815 cells, mouse RBL-2H3 cells, human HL-60 cells, human NAMALWA cells,human Macrophage cells, mouse RAW 264.7 cells, human KG-1 cells, mouseM1 cells, human PBMC cells, mouse BW5147 (T200-A)5.2 cells, humanCCRF-CEM cells, mouse EL4 cells, human Jurkat cells, human SCID.adhcells, human U-937 cells or any combination of cells thereof.

The term “primary cells” and its grammatical equivalents, as usedherein, can refer to cells taken directly from an organism, typicallyliving tissue of a multicellular organism, such as animals or plants. Inmany cases, primary cells may be established for growth in vitro. Insome cases, primary cells may be just removed from the organism and havenot been established for growth in vitro yet before the transfection. Insome embodiments, the primary cells can also be expanded in vitro, i.e.primary cells may also include progeny cells that are generated fromproliferation of the cells taken directly from an organism. In thesecases, the progeny cells do not exhibit the indefinite proliferativeproperty as cells in established cell lines. For instance, the hostcells may be human primary T cells, while prior to the transfection, theT cells have been exposed to stimulatory factor(s) that may result in Tcell proliferation and expansion of the cell population.

The cells to be genetically modified may be primary cells from tissuesor organs, such as, but not limited to, brain, lung, liver, heart,spleen, pancreas, small intestine, large intestine, skeletal muscle,smooth muscle, skin, bones, adipose tissues, hairs, thyroid, trachea,gall bladder, kidney, ureter, bladder, aorta, vein, esophagus,diaphragm, stomach, rectum, adrenal glands, bronchi, ears, eyes, retina,genitals, hypothalamus, larynx, nose, tongue, spinal cord, or ureters,uterus, ovary, testis, and any combination thereof. In certainembodiments, the cells may include, but not limited to, hematocyte,trichocyte, keratinocyte, gonadotrope, corticotrope, thyrotrope,somatotrope, lactotroph, chromaffin cell, parafollicular cell, glomuscell, melanocyte, nevus cell, merkel cell, odontoblast, cementoblast,corneal keratocyte, retina muller cell, retinal pigment epithelium cell,neuron, glia, ependymocyte, pinealocyte, pneumocyte, clara cell, gobletcell, G cell, D cell, Enterochromaffin-like cell, gastric chief cell,parietal cell, foveolar cell, K cell, D cell, I cell, paneth cell,enterocyte, microfold cell, hepatocyte, hepatic stellate cell,cholecystocyte, centroacinar cell, pancreatic stellate cell, pancreaticα cell, pancreatic β cell, pancreatic δ cell, pancreatic F cell,pancreatic c cell, thyroid parathyroid, oxyphil cell, urothelial cell,osteoblast, osteocyte, chondroblast, chondrocyte, fibroblast, fibrocyte,myoblast, myocyte, myosatellite cell, tendon cell, cardiac muscle cell,lipoblast, adipocyte, interstitial cell of cajal, angioblast,endothelial cell, mesangial cell, juxtaglomerular cell, macula densacell, stromal cell, interstitial cell, telocyte, simple epithelial cell,podocyte, kidney proximal tubule brush border cell, sertoli cell, leydigcell, granulosa cell, peg cell, germ cell, spermatozoon ovum,lymphocyte, myeloid cell, endothelial progenitor cell, endothelial stemcell, angioblast, mesoangioblast, pericyte mural cell, and anycombination thereof. In many instances, the cell to be modified may be astem cell, such as, but not limited to, embryonic stem cell,hematopoietic stem cell, epidermal stem cell, epithelial stem cell,bronchoalveolar stem cell, mammary stem cell, mesenchymal stem cell,intestine stem cell, endothelial stem cell, neural stem cell, olfactoryadult stem cell, neural crest stem cell, testicular cell, and anycombination thereof. Sometimes, the cell can be an induced pluripotentstem cell that is derived from any type of tissue.

In some embodiments, the cell to be genetically modified may be amammalian cell. In some embodiments, the cell may be an immune cell.Non-limiting examples of the cell can include a B cell, a basophil, adendritic cell, an eosinophil, a gamma delta T cell, a granulocyte, ahelper T cell, a Langerhans cell, a lymphoid cell, an innate lymphoidcell (ILC), a macrophage, a mast cell, a megakaryocyte, a memory T cell,a monocyte, a myeloid cell, a natural killer T cell, a neutrophil, aprecursor cell, a plasma cell, a progenitor cell, a regulatory T-cell, aT cell, a thymocyte, any differentiated or de-differentiated cellthereof, or any mixture or combination of cells thereof. In certaincases, the cell may be a T cell. In some embodiments, the cell may be aprimary T cell. In certain cases, the cell may be an antigen-presentingcell (APC). In some embodiments, the cell may be a primary APC. The APCsin connection with the present disclosure may be a dendritic cell,macrophage, B cell, other non-professional APCs, or any combinationthereof.

In some embodiments, the cell may be an ILC (innate lymphoid cell), andthe ILC can be a group 1 ILC, a group 2 ILC, or a group 3 ILC. Group 1ILCs may generally be described as cells controlled by the T-bettranscription factor, secreting type-1 cytokines such as IFN-gamma andTNF-alpha in response to intracellular pathogens. Group 2 ILCs maygenerally be described as cells relying on the GATA-3 and ROR-alphatranscription factors, producing type-2 cytokines in response toextracellular parasite infections. Group 3 ILCs may generally bedescribed as cells controlled by the ROR-gamma t transcription factor,and produce IL-17 and/or IL-22.

In some embodiments, the cell may be a cell that is positive or negativefor a given factor. In some embodiments, a cell may be a CD3+ cell, CD3−cell, a CD5+ cell, CD5− cell, a CD7+ cell, CD7− cell, a CD14+ cell,CD14− cell, CD8+ cell, a CD8− cell, a CD103+ cell, CD103− cell, CD11b+cell, CD11b− cell, a BDCA1+ cell, a BDCA1− cell, an L-selectin+ cell, anL-selectin− cell, a CD25+, a CD25− cell, a CD27+, a CD27− cell, a CD28+cell, CD28− cell, a CD44+ cell, a CD44− cell, a CD56+ cell, a CD56−cell, a CD57+ cell, a CD57− cell, a CD62L+ cell, a CD62L− cell, a CD69+cell, a CD69− cell, a CD45RO+ cell, a CD45RO− cell, a CD127+ cell, aCD127− cell, a CD132+ cell, a CD132− cell, an IL-7+ cell, an IL-7− cell,an IL-15+ cell, an IL-15− cell, a lectin-like receptor G1 positive cell,a lectin-like receptor G1 negative cell, or an differentiated orde-differentiated cell thereof. The examples of factors expressed bycells is not intended to be limiting, and a person having skill in theart will appreciate that the cell may be positive or negative for anyfactor known in the art. In some embodiments, the cell may be positivefor two or more factors. For example, the cell may be CD4+ and CD8+. Insome embodiments, the cell may be negative for two or more factors. Forexample, the cell may be CD25−, CD44−, and CD69−. In some embodiments,the cell may be positive for one or more factors, and negative for oneor more factors. For example, a cell may be CD4+ and CD8−.

It should be understood that cells used in any of the methods disclosedherein may be a mixture (e.g., two or more different cells) of any ofthe cells disclosed herein. For example, a method of the presentdisclosure may comprise cells, and the cells are a mixture of CD4+ cellsand CD8+ cells. In another example, a method of the present disclosuremay comprise cells, and the cells are a mixture of CD4+ cells and naïvecells.

As provided herein, the transposase and the transposon can be introducedin to a cell through a number of approaches. The term “transfection” andits grammatical equivalents as used herein can generally refer to aprocess whereby nucleic acids are introduced into eukaryotic cells. Thetransfection methods that can be used in connection with the subjectmatter can include, but not limited to, electroporation, microinjection,calcium phosphate precipitation, cationic polymers, dendrimers,liposome, microprojectile bombardment, fugene, direct sonic loading,cell squeezing, optical transfection, protoplast fusion, impalefection,magnetofection, nucleofection, or any combination thereof. In manycases, the transposase and transposon described herein can betransfected into a host cell through electroporation. Sometimes,transfection can also be done through a variant of electroporationmethod, such as nucleofection (also known as Nucleofector™ technology).The term “electroporation” and its grammatical equivalents as usedherein can refer to a process whereby an electrical field is applied tocells in order to increase the permeability of the cell membrane,allowing chemicals, drugs, or DNA to be introduced into the cell. Duringelectroporation, the electric filed is often provided in the form of“pulses” of very brief time periods, e.g. 5 milliseconds, 10milliseconds, and 50 milliseconds. As understood by those skilled in theart, electroporation temporarily opens up pores in a cell's outermembrane by use of pulsed rotating electric fields. Methods andapparatus used for electroporation in vitro and in vivo are also wellknown. Various electric parameters can be selected dependent on the celltype being electroporated and physical characteristics of the moleculesthat are to be taken up by the cell, such as pulse intensity, pulselength, number of pulses).

Applications

The subject matter, e.g., the compositions (e.g., mutant TcBustertransposases, fusion transposases, TcBuster transposons), systems andmethods, provided herein may find use in a wide range of applicationsrelating to genome editing, in various aspects of modern life.

Under certain circumstances, advantages of the subject matter describedherein may include, but not limited to, reduced costs, regulatoryconsideration, lower immunogenicity and less complexity. In some cases,a significant advantage of the present disclosure is the hightransposition efficiency. Another advantage of the present disclosure,in many cases, is that the transposition system provided herein can be“tunable”, e.g., transposition can be designed to target select genomicregion rather than random insertion.

One non-limiting example is related to create genetically modified cellsfor research and clinical applications. For example, as discussed above,genetically modified T cells can be created using the subject matterprovided herein, which may find use in helping people fighting against avariety of diseases, such as, but not limited to, cancer and infectiousdisease.

One particular example includes generation of genetically modifiedprimary leukocytes using the methods provided herein, and administeringthe genetically modified primary leukocytes to a patient in needthereof. The generation of genetically modified primary leukocytes caninclude introducing into a leukocyte a transposon and a mutant TcBustertransposase or the fusion transposase as described herein, which canrecognize the transposon, thereby generating a genetically modifiedleukocyte. In many cases, the transposon may comprise a transgene. Thetransgene can be a cellular receptor, an immunological checkpointprotein, a cytokine, and any combination thereof. Sometimes, a cellularreceptor can include, but not limited to a T cell receptor (TCR), a Bcell receptor (BCR), a chimeric antigen receptor (CAR), or anycombination thereof. In some other cases, the transposon and thetransposase are designed to delete or modify an endogenous gene, forinstance, a cytokine, an immunological checkpoint protein, an oncogene,or any combination thereof. The genetic modification of the primaryleukocytes can be designed to facilitate immunity against an infectiouspathogen or cancer cells that render the patient in diseased state.

Another non-limiting example is related to create genetically modifiedorganisms for agriculture, food production, medicine, and pharmaceutics.The species that can be genetically modified span a wide range,including, but not limited to, plants and animals. The geneticallymodified organisms, such as genetically modified crops or livestock, maybe modified in a certain aspect of their physiological properties.Examples in food crops include resistance to certain pests, diseases, orenvironmental conditions, reduction of spoilage, or resistance tochemical treatments (e.g. resistance to a herbicide), or improving thenutrient profile of the crop. Examples in non-food crops includeproduction of pharmaceutical agents, biofuels, and other industriallyuseful goods, as well as for bioremediation. Examples in livestockinclude resistance to certain parasites, production of certain nutritionelements, increase in growth rate, and increase in milk production.

The term “about” and its grammatical equivalents in relation to areference numerical value and its grammatical equivalents as used hereincan include a range of values plus or minus 10% from that value. Forexample, the amount “about 10” includes amounts from 9 to 11. The term“about” in relation to a reference numerical value can also include arange of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%from that value.

EXAMPLES

The examples below further illustrate the described embodiments withoutlimiting the scope of this disclosure.

Example 1. Materials and Methods

This example describes several methods utilized for generation andevaluation of exemplary mutant TcBuster transposases.

Site Directed Mutagenesis for TcBuster Mutant Preparation

Putative hyperactive TcBuster (TcB) transposase mutants were identifiedby nucleotide sequence and amino acid alignment of hAT and bustersubfamilies. The Q5 site-directed mutagenesis kit (New England BioLabs)was used for all site-directed mutagenesis. Following PCR mutagenesis,PCR products were purified with GeneJET PCR purification kit (ThermoFisher Scientific). A 20 uL ligation reaction of purified PCR productswas performed using T4 DNA ligase (New England BioLabs). 5 uL ofligation reaction was used for transformation in DH10Beta cells. Directcolony sequencing through Sequetech was used to confirm the presence ofdesired mutations. DNA for confirmed mutations was prepped usingZymoPURE plasmid miniprep kits (Zymo Research).

Measuring Transection Efficiency in HEK-293T Cells

HEK-293T cells were plated at 300,000 cells per well of a 6 well plateone day prior to transfection. Cells were transfected with 500 ngtransposon carrying mCherry-puromycin cassette and 62.5 ng TcBtransposase using TransIT X2 reagent per manufacturer's instructions(Mirus Bio). Two days post-transfection, cells were re-plated withpuromycin (1 ug/mL) at a density of 3,000 cells/well of a 6 well platein triplicate in DMEM complete media, or re-plated without puromycinselection. Stable integration of the transgene was assessed by colonycounting of puromycin treated cells (each cell that survived drugselection formed a colony) or flow cytometry. For colony counting, twoweeks post-puromycin selection, DMEM complete+puromycin media wasremoved. Cells were washed with 1×PBS and cells were stained with 1×crystal violet solution for 10 minutes. Plates were washed twice withPBS and colonies counted.

For flow cytometry analysis, stable integration of the transgene wasassessed by detection of mCherry fluorescence in cells grown withoutdrug selection. Transfected cells were harvested at indicated timepoints post-transfection, washed 1× with PBS and resuspended in 200 uLRDFII buffer for analysis. Cells were analyzed using Novocyte (AceaBiosciences) and mCherry expression was assessed using the PE-Texas redchannel.

Screening of TcB Transposase Mutants in HEK-293T Cells

HEK-293T cells were plated at 75,000 cells per well of a 24 well plateone day prior to transfection. Cells were transfected with 500 ngtransposon and 125 ng transposase using TransIT X2 reagent in duplicateper manufacturer's instructions (Mirus Bio). Stable integration of thetransgene was assessed by detection of mCherry fluorescence. Cells wereharvested at 14 days post-transfection, washed 1× with PBS andresuspended in 200 uL RDFII buffer. Cells were analyzed using Novocyte(Acea Biosciences) and mCherry expression was assessed using thePE-Texas red channel.

Transfection of TcBuster Transposon and Transposase in CD3+ T-Cells

CD3+ T-cells were enriched and cryopreserved from leukopaks(StemCellTechnologies). CD3+ T-cells were thawed and activated usingCD3/CD28 Dynabeads (ThermoFisher) for 2 days in X-Vivo-15 mediasupplemented with human serum and IL-2, IL-15 and IL-7 cytokines. Priorto transfection, CD3/CD28 beads were removed, cells washed andelectroporated using Neon Transfection system (ThermoFisher) withTcBuster transposon (mini-circle carrying TcBuster and Sleeping beautyIR/DRs and GFP cargo) and TcBuster or Sleeping Beauty transposases inRNA form. As a viability control, cells were “pulse” electroporatedwithout DNA or RNA. Electroporated cells were expanded for 21 dayspost-transfection and viability stable integration of GFP cargo wasassessed by flow cytometry. Viability was measured by SSC-A vs FSC-A andstandardized to pulse only control, and GFP expression was assessedusing FITC channel on days 2, 7, 14 and 21.

Example 2. Exemplary Transposon Constructs

The aim of this study was to examine transposition efficiency ofdifferent exemplary TcBuster transposon constructs. Inventors compared10 TcBuster (TcB) transposon (Tn) configurations (FIG. 1A) to test theirtransposition efficiency in mammalian cells. These 10 TcB Tns differedin the promoter used (EFS vs CMV), IR/DR sequence and direction of thetransposon cargo. The transposons each contained an identical cassettecoding for mCherry linked by 2A to a drug-resistance gene, puromycin, sothat transfected cells could be identified by fluorescence and/orselection with puromycin. HEK-293T cells were transfected with one ofthe 10 TcB Tns and TcB wild-type transposase (ratio of 1 transposon:1transposase). Stable integration of the transgene was assessed by flowcytometry by detection of mCherry fluorescence for 10-30 dayspost-transfection (FIG. 1B).

It was found that, under experimental conditions, stable expression ofthe transgene mCherry was greatly enhanced using the CMV promotercompared to EFS. Transposition appeared to only occur when sequence 1IR/DRs was used. It was also found that transcription of the cargo inthe reverse direction promoted greater transposition activity comparedto the forward direction.

TcB Tn-8 showed the greatest transposition efficiency among the test 10Tns by flow cytometry. To confirm the transposition efficiency of TcBTn-8, HEK-293T cells were transfected with TcB Tn-8 with WT transposaseor V596A mutant transposase. Two days post-transfection, cells werere-plated with puromycin (1 ug/mL) at a density of 3,000 cells/well of a6 well plate in triplicate in DMEM complete media. After selection fortwo weeks, each cell that survived drug selection formed a colony, whichwas assessed for mCherry expression (FIG. 3A) and counted to confirmstable integration of the transgene (FIGS. 3B-C). Transpositionefficiency of TcB-Tn 8 was confirmed by expression of mCherry andpuromycin resistant colonies in HEK-293T cells.

Example 3. Exemplary Transposase Mutants

The aim of this study was to generate TcBuster transposase mutants andexamine their transposition efficiency.

To this end, inventors have generated a consensus sequence by comparingcDNA and amino acid sequences of wild-type TcB transposase to othersimilar transposases. For the comparison, sleeping beauty wasresurrected by the alignment of 13 similar transposases and SPIN by thealignment of SPIN like transposases from 8 separate organisms. SPIN andTcBuster are a part of the abundant hAT family of transposases.

The hAT transposon family consists of two subfamilies: AC, such as hashobo, hermes, and Tol2, and the Buster subfamily, such as SPIN andTcBuster. Amino acid sequence of TcBuster was aligned to amino acidsequences of both AC and Buster subfamily members to identify key aminoacids that are not conserved in TcBuster that may be targets ofhyperactive substitutions. Alignment of TcBuster to the AC subfamilymembers Hermes, Hobo, Tag2, Tam3, Herves, Restless, and Tol2 allowed usto identify amino acids within areas of high conservation that could besubstituted in TcBuster (FIG. 4). Further, sequence alignment ofTcBuster to the Buster subfamily led to a larger number of candidateamino acids that may be substituted (FIG. 5). Candidate TcB transposasemutants were generated using oligonucleotides comprising site mutationsas listed in Table 7. The mutants were then sequence verified, clonedinto pCDNA-DEST40 expression vector (FIG. 6) and mini-prepped prior totransfection.

TABLE 7 SEQ Amino Acid ID Substitutions Oligo NameOligonucleotide Sequence (5′-3′) NO Q82E TCB Q82E GATTTGCGAGgAGGTAGTCAAC14 FWD Q82E TCB Q82E ACACAAAGTCCGTTGGGC 15 REV A358E TCBA358ECGCGTCTTCGaaTTGCTGTGTGAC 16 FWD A358E TCBA358E CGCATTCAACGGCCGAGA 17 REVA358S TCBA358S GCGCGTCTTCagTTTGCTGTGTGACG 18 FWD A358S TCBA358SGCATTCAACGGCCGAGAC 19 REV A358K TCBA358K GCGCGTCTTCaagTTGCTGTGTGACG 20FWD A358K TCBA358K GCATTCAACGGCCGAGAC 21 REV S447E TCBS447ECAAGGTAAATgagCGCATTAACAGTATTAAATC 22 FWD S447E TCBS447EAAGATTGTGCTATTCGGC 23 REV I452F TCBI452F CATTAACAGTtTTAAATCAAAGTTGAAG 24FWD I452F TCBI452F CGGCTATTTACCTTGAAG 25 REV N281E TCBN281ECATCCCATGGgaaCTGTGTTACC 26 FWD N281E TCBN281E GAGTGCTTTTCGAAATAGG 27 REVI223Q TCBI223Q CGGTCTTGCAcagCTGCTTGTGTTTG 28 FWD I223Q TCBI223QGCAACATCTGTTGACTCG 29 REV P510D TCBP510DGTATTTTCCAgatACGTGTAATAATATCTCCTG 30 FWD P510D TCBP510DTCCAGAAAGGTGTTCTTAAG 31 REV P510N TCBP510NGTATTTTCCAaatACGTGTAATAATATCTCC 32 FWD P510N TCBP510NTCCAGAAAGGTGTTCTTAAG 33 REV E517R TCBE517R CTCCTGGGTGcggAATCCTTTCAATG 34FWD E517R TCBE517R ATATTATTACACGTAGGTGG 35 REV K590T TCBK590TGAAATTAGCAcACGAGCTGTC 36 FWD K590T TCBK590T TGGAAATTCGTCCATCAG 37 REVN885S TCBN885S GCAGGTAGTCagcAATTCCTCAC 38 FWD N885S TCBN885STCGCAAATCACACAAAGTC 39 REV S109D TCBS109DTAAAGGCAAGgacGAATACTTCAAAAGAAAATGTAAC 40 FOR S109D TCBS109DTAAAGGCAAGgacGAATACTTCAAAAGAAAATGTAAC 41 REV K135E TCBK135EGGACGATAACgagAACCTCCTGA 42 FWD K135E TCBK135E CTTACGTATCGCTCAAAAGTATG 43REV D99A TcB-D99A F ACGCCATTTGgcaACAAAGCATC 44 D99A TcB-D99A RTTCAGTTTGGCCGGGTTA 45 D132A TcB-D132A-F ATACGTAAGGgcaGATAACAAGAACC 46D132A TcB-D132A-R CGCTCAAAAGTATGCTTC 47 E159A TcB-E159A-FTACCATAGCGgcgAAGTTGATCAAG 48 E159A TcB-E159A-R TATGCCTCGCCCTGTTTA 49D189A TcB-D189A-F CCCCCTGTCCgcaACGACTATTTC 50 D189A TcB-D189A-RACGAGATCAACTTTGCTC 51 D227A TcB-D227A-F CGAGTCAACAgcaGTTGCCGGTC 52 D227ATcB-D227A-R TCCATCTGCAGCGTAAAC 53 E243A TcB-E243A-FGTACATACATgcaAGCTCTTTTG 54 E243A TcB-E243A-R CTAACAAACACAAGCAGG 55 V377TTcB-V377T-F TCATACCGAAacgAGGTGGCTGTC 56 V377T TcB-V377T-RAGAAGAAGATTTTTATGCAGG 57 S225W TcB-S225W-F GATGGACGAGtggACAGATGTTGC 58S225W TcB-S225W-R TGCAGCGTAAACCCACAT 59 Y155F TcB-Y155F-FGGGCGAGGCAtttACCATAGCGG 60 Y155F TcB-Y155F-RTGTTTAGCTATTCTCAAACTGACGAGATAAG 61 D132A TcB-D132A-FATACGTAAGGgcaGATAACAAGAACC 62 D132A TcB-D132A-R CGCTCAAAAGTATGCTTC 63E159A TcB-E159A-F TACCATAGCGgcgAAGTTGATCAAG 64 E159A TcB-E159A-RTATGCCTCGCCCTGTTTA 65 D189A TcB-D189A-F CCCCCTGTCCgcaACGACTATTTC 66D189A TcB-D189A-R ACGAGATCAACTTTGCTC 67 D227A TcB-D227A-FCGAGTCAACAgcaGTTGCCGGTC 68 D227A TcB-D227A-R TCCATCTGCAGCGTAAAC 69 E243ATcB-E243A-F GTACATACATgcaAGCTCTTTTG 70 E243A TcB-E243A-RCTAACAAACACAAGCAGG 71 V377T TcB-V377T-F TCATACCGAAacgAGGTGGCTGTC 72V377T TcB-V377T-R AGAAGAAGATTTTTATGCAGG 73 S224W TcB-S224W-FGATGGACGAGtggACAGATGTTGC 74 S224W TcB-S224W-R TGCAGCGTAAACCCACAT 75Y155F TcB-Y155F-F GGGCGAGGCAtttACCATAGCGG 76 Y155F TcB-Y155F-RTGTTTAGCTATTCTCAAACTGACGAGATAAG 77

To examine the transposition efficiency of the TcB transposase mutants,HEK-293T cells were transfected with TcB Tn-8 (mCherry-puromycincassette) with WT transposase or V596A mutant transposase, or thecandidate transposase mutants in duplicate. Cells were grown in DMEMcomplete (without drug selection) and mCherry expression was assessed byflow cytometry on Day 14 post-transfection. Over 20 TcB transposasemutants were identified that had transposition efficiency greater thanthe wild-type transposase (FIG. 7). It was discovered that among theseexamined mutants, one mutant transposase containing a combination ofthree amino acid substitutions, D189A, V377T, and E469K, led to asubstantial increase in transposition activity, as compared to mutantscontaining respective single substitutions. Mutants with hightransposition activity also included, among others, K573E/E578L, I452F,A358K, V297K, N85S, S447E, E247K, and Q258T.

Among these examined mutants, it was discovered that most ofsubstitutions to a positively charged amino acid, such as Lysine (K) orArginine (R), in proximity to one of the catalytic triad amino acids(D234, D289, and E589) increased transposition. In addition, removal ofa positive charge, or addition of a negative charge decreasedtransposition. These data suggests that amino acids close to thecatalytic domain may help promote the transposition activity of TcB, inparticular, when these amino acids are mutated to positively chargedamino acids.

The amino acid sequence of the hyperactive TcBuster mutantD189A/V377T/E469K (SEQ ID NO: 78) is illustrated in FIG. 12. Furthermutational analysis of this mutant will be performed. As illustrated inFIG. 13, the TcBuster mutant D189A/V377T/E469K/I452F (SEQ ID NO: 79)will be constructed. As illustrated in FIG. 14, the TcBuster mutantD189A/V377T/E469K/N85S (SEQ ID NO: 80) will be constructed. Asillustrated in FIG. 15, the Tc Buster mutant D189A/V377T/E469K/S358K(SEQ ID NO: 81) will be constructed. As illustrated in FIG. 16, the TcBuster mutant D189A/V377T/E469K/K573E/E578L (SEQ ID NO: 13) will beconstructed. In each of FIGS. 12-16, the domains of TcBuster areindicated as follows: ZnF-BED (lowercase lettering), DNABinding/oligomization domain (bold lettering), catalytic domain(underlined lettering), and insertion domain (italicized lettering); thecore D189A/V377T/E469K substitutions are indicated in larger, bold,italicized, and underlined letters; and the additional substitutions areindicated in large, bold letters. Each of these constructs will betested as already described and are anticipated to show hyperactivity incomparison to the wild type TcBuster.

Example 4. Exemplary Fusion Transposase Containing Tag

The aim of this study was to generate and examine the transpositionefficiency of fusion TcBuster transposases. As an example, protein tag,GST or PEST domain, was fused to N-terminus of TcBuster transposase togenerate fusion TcBuster transposases. A flexible linker GGSGGSGGSGGSGTS(SEQ ID NO: 9), which was encoded by SEQ ID NO: 10, was used to separatethe GST domain/PEST domain from TcBuster transposase. The presence ofthis flexibility linker may minimize non-specific interaction in thefusion protein, thus increasing its activity. The exemplary fusiontransposases were transfected with TcB Tn-8 as described above andtransposition efficiency was measured by mCherry expression on Day 14 byflow cytometry. Transposition efficiency was not affected by tagging ofGFP or PEST domain (FIG. 9), suggesting that fusing the transposase DNAbinding domains to direct integration of TcBuster cargo to selectgenomic sites, such as safe harbor sites, could be a viable option forTcBuster allowing for a safer integration profile.

Example 5. Exemplary Fusion Transposase Comprising Tale Domain

The aim of this study is to generate a fusion TcBuster transposasecomprising a TALE domain and to examine the transposition activity ofthe fusion transposase. A TALE sequence (SEQ ID NO: 11) is designed totarget human AAVS1 (hAAVS1) site of human genome. The TALE sequence isthus fused to N-terminus of a wild-type TcBuster transposase (SEQ IDNO: 1) to generate a fusion transposase. A flexible linker Gly4Ser2 (SEQID NO: 88), which is encoded by SEQ ID NO: 12, is used to separate theTALE domain and the TcBuster transposase sequence. The exemplary fusiontransposase has an amino acid sequence SEQ ID NO: 8.

The exemplary fusion transposase will be transfected with a TcB Tn-8 asdescribed above into Hela cells with the aid of electroporation. The TcBTn-8 comprises a reporter gene mCherry. The transfection efficiency canbe examined by flow cytometry 2 days post-transfection that countsmCherry-positive cells. Furthermore, next-generation sequencing will beperformed to assess the mCherry gene insertion site in the genome. It isexpected that the designed TALE sequence can mediate the targetinsertion of the mCherry gene at a genomic site near hAAVS1 site.

Example 6. Transposition Efficiency in Primary Human T-Cells

The aim of this study was to develop TcBuster transposon system toengineer primary CD3+ T cells. To this end, inventors incorporated anexemplary TcBuster transposon carrying a GFP transgene into amini-circle plasmid. Activated CD3+ T cells were electroporated with TcBmini-circle transposon and RNA transposases, such as WT TcBustertransposase, and select exemplary mutants as described in Example 2. Thetransgene expression was monitored for 21 days post-electroporation byflow cytometry.

It was found that transposition of the TcB transposon was improvednearly two folds using the exemplary mutants, V377T/E469K andV377T/E469K/D189A, 14 days post-transfection compared to the WT TcBustertransposase and V596A mutant transposase (FIG. 10A). Further, meantransposition efficiency with the hyperactive mutants V377T/E469K andV377T/E469K/D189A was two (mean=20.2) and three (mean=24.1) times moreefficient compared to SB11 (mean=8.4), respectively.

Next, the viability of CD3+ T cells was assessed two dayspost-electroporation with the mini-circle TcB transposon and RNAtransposase. It was found that viability was moderately decreased whenCD3+ T-cells were transfected with TcB mini-circle and RNA transposase;however, the cells quickly recovered viability by Day 7 (FIG. 10B).These experiments demonstrate the competency of the TcBuster transposonsystem, according to some embodiments of the present disclosure, incellular engineering of primary T cells.

Example 7. Generation of Chimeric Antigen Receptor-Modified T Cells forTreatment of Cancer Patient

A mini-circle plasmid containing aforementioned TcB Tn-8 construct canbe designed to harbor a chimeric antigen receptor (CAR) gene between theinverted repeats of the transposon. The CAR can be designed to havespecificity for the B-cell antigen CD19, coupled with CD137 (acostimulatory receptor in T cells [4-1BB]) and CD3-zeta (asignal-transduction component of the T-cell antigen receptor) signalingdomains.

Autologous T cells will be obtained from peripheral blood of a patientwith cancer, for example, leukemia. The T cells can be isolated bylysing the red blood cells and depleting the monocytes by centrifugationthrough a PERCOLL™ gradient. CD3+ T cells can be isolated by flowcytometry using anti-CD3/anti-CD28-conjugated beads, such as DYNABEADM-450 CD3/CD28T. The isolated T cells will be cultured under standardconditions according to GMP guidance.

Genetic modification of the primary T cells will be conducted using amutant TcBuster transposase (SEQ ID NO: 13) comprising amino acidsubstitutions V377T, E469K, D189A, K573E and E578L and the TcBuster Tn-8transposase comprising the CAR, as described above. The T cells will beelectroporated in the presence of the mutant TcBuster transposase andthe CAR-containing Tn-8 transposase. Following transfection, T cellswill be treated with immunostimulatory reagents (such as anti-CD3antibody and IL-2, IL-7, and IL-15) for activation and expansion.Validation of the transfection will be performed by next-generationsequencing 2 weeks post-transfection. The transfection efficiency andtransgene load in the transfected T cells can be determined to assistthe design of treatment regimen. Certain measure will also be taken toeliminate any safety concern if risky transgene insertion site isuncovered by the sequencing results.

Infusion of the chimeric antigen receptor modified T cells (CAR-T cells)back to the cancer patient will start after validation of transgeneinsertion and in vitro expansion of the CAR-T cells to a clinicallydesirable level.

The infusion dose will be determined by a number of factors, including,but not limited to, the stage of the cancer, the treatment history ofthe patient, and the CBC (complete blood cell count) and vital signs ofthe patient on the day of treatment. Infusion dose may be escalated ordeescalated depending on the progression of the disease, the repulsionreaction of the patient, and many other medical factors. In themeantime, during the treatment regimen, quantitativepolymerase-chain-reaction (qPCR) analysis will be performed to detectchimeric antigen receptor T cells in blood and bone marrow. The qPCRanalysis can be utilized to make medical decision regarding the dosingstrategy and other treatment plans.

TABLE 8 Amino Acid and Nucleotide Sequences Sequence DescriptionAmino Acid Sequence Or Nucleotide Sequence (SEQ ID NO)Wild-type TcBuster (accession number: ABF20545) transposaseMMLNWLKSGKLESQSQEQSSCYLENSNCLPPTLDSTDIIGEENKAGTTSRKKRKYDEDYLNFGFTWTGDKDEPNGLCVICEQVVNNSSLNPAKLKRHLDTKHPTLKGKSEYFKRKCNELNQKKHTFERYVRDDNKNLLKASYLVSLRIAKQGEAYTIAEKLIKPCTKDLTTCVFGEKFASKVDLVPLSDTTISRRIEDMSYFCEAVLVNRLKNAKCGFTLQMDESTDVAGLAILLVFVRYIHESSFEEDMLFCKALPTQTTGEEIFNLLNAYFEKHSIPWNLCYHICTDGAKAMVGVIKGVIARIKKLVPDIKASHCCLHRHALAVKRIPNALHEVLNDAVKMINFIKSRPLNARVFALLCDDLGSLHKNLLLHTEVRWLSRGKVLTRFWELRDEIRIFFNEREFAGKLNDTSWLQNLAYIADIFSYLNEVNLSLQGPNSTIFKVNSRINSIKSKLKLWEECITKNNTECFANLNDFLETSNTALDPNLKSNILEHLNGLKNTFLEYFPPTCNNISWVENPFNECGNVDTLPIKEREQLIDIRTDTTLKSSFVPDGIGPFWIKLMDEFPEISKRAVKELMPFVTTYLCEKSFSVYVATKTKYRNRLDAEDDMRLQLTTIHPDIDNLCNNKQAQKSH (SEQ ID NO: 1)Wild-type TcBusteratgatgttgaattggctgaaaagtggaaagcttgaaagtcaatcacaggaacagagtt transposasecctgctaccttgagaactctaactgcctgccaccaacgctcgattctacagatattatcggtgaagagaacaaagctggtaccacctctcgcaagaagcggaaatatgacgaggactatctgaacttcggttttacatggactggcgacaaggatgagcccaacggactttgtgtgatttgcgagcaggtagtcaacaattcctcacttaacccggccaaactgaaacgccatttggacacaaagcatccgacgcttaaaggcaagagcgaatacttcaaaagaaaatgtaacgagctcaatcaaaagaagcatacttttgagcgatacgtaagggacgataacaagaacctcctgaaagcttcttatctcgtcagtttgagaatagctaaacagggcgaggcatataccatagcggagaagttgatcaagccttgcaccaaggatctgacaacttgcgtatttggagaaaaattcgcgagcaaagttgatctcgtccccctgtccgacacgactatttcaaggcgaatcgaagacatgagttacttctgtgaagccgtgctggtgaacaggttgaaaaatgctaaatgtgggtttacgctgcagatggacgagtcaacagatgttgccggtcttgcaatcctgcttgtgtttgttaggtacatacatgaaagctcttttgaggaggatatgttgttctgcaaagcacttcccactcagacgacaggggaggagattttcaatcttctcaatgcctatttcgaaaagcactccatcccatggaatctgtgttaccacatttgcacagacggtgccaaggcaatggtaggagttattaaaggagtcatagcgagaataaaaaaactcgtccctgatataaaagctagccactgttgcctgcatcgccacgctttggctgtaaagcgaataccgaatgcattgcacgaggtgctcaatgacgctgttaaaatgatcaacttcatcaagtctcggccgttgaatgcgcgcgtcttcgctttgctgtgtgacgatttggggagcctgcataaaaatcttcttcttcataccgaagtgaggtggctgtctagaggaaaggtgctgacccgattttgggaactgagagatgaaattagaattttcttcaacgaaagggaatttgccgggaaattgaacgacaccagttggttgcaaaatttggcatatatagctgacatattcagttatctgaatgaagttaatctttccctgcaagggccgaatagcacaatcttcaaggtaaatagccgcattaacagtattaaatcaaagttgaagttgtgggaagagtgtataacgaaaaataacactgagtgttttgcgaacctcaacgattttttggaaacttcaaacactgcgttggatccaaacctgaagtctaatattttggaacatctcaacggtcttaagaacacctttctggagtattttccacctacgtgtaataatatctcctgggtggagaatcctttcaatgaatgcggtaacgtcgatacactcccaataaaagagagggaacaattgattgacatacggactgatacgacattgaaatcttcattcgtgcctgatggtataggaccattctggatcaaactgatggacgaatttccagaaattagcaaacgagctgtcaaagagctcatgccatttgtaaccacttacctctgtgagaaatcattttccgtctatgtagccacaaaaacaaaatatcgaaatagacttgatgctgaagacgatatgcgactccaacttactactatccatccagacattgacaacctttgtaacaacaagcaggctcagaaatcccactga (SEQ ID NO: 2)IRDR-L-Seq1 Cagtgttcttcaacctttgccatccggcggaaccctttgtcgagatatttttttttatggaacccttcatttagtaatacacccagatgagattttagggacagctgcgttgacttgttacgaacaaggtgagcccgtgctttggtctagccaagggcatggtaaagactatattcgcggcgttgtgacaatttaccgaacaactccgcggccgggaagccgatctcggcttgaacgaattgttaggtggcggtacttgggtcgatatcaaagtgcatcacttcttcccgtatgcccaactttgtatagagagccactgcgggatcgtcaccgtaatctgcttgcacgtagatcacataagcaccaagcgcgttggcctcatgcttgaggagattgatgagcgcggtggcaatgccctgcctccggtgctcgccggagactgcgagatcatagatata (SEQ ID NO: 3)IRDR-R-Seq1 >gatatcaagcttatcgataccgtcgacctcgagatttctgaacgattctaggttaggatcaaacaaaatacaatttattttaaaactgtaagttaacttacctttgcttgtctaaaccaaaaacaacaacaaaactacgaccacaagtacagttacatatttttgaaaattaaggttaagtgcagtgtaagtcaactatgcgaatggataacatgtttcaacatgaaactccgattgacgcatgtgcattctgaagagcggcgcggccgacgtctctcgaattgaagcaatgactcgcggaaccccgaaagcctttgggtggaaccctagggttccgcggaacacaggttgaagaacactg (SEQ ID NO: 4) IRDR-L-Seq2Cctgcaggagtgttcttcaacctttgccatccggcggaaccctttgtcgagatatttttttttatggaacccttcatttagtaatacacccagatgagattttagggacagctgcgttgacttgttacgaacaaggtgagcccgtgctttggtaataaaaactctaaataagatttaaatttgcatttatttaaacaaactttaaacaaaaagataaatattccaaataaaataatatataaaataaaaaataaaaattaatgacttttttgcgcttgcttattattgcacaaattatcaatatcgggatggatcgttgtttttt (SEQ ID NO: 5) IRDR-R-Seq2Gagccaattcagcatcatatttctgaacgattctaggttaggatcaaacaaaatacaatttattttaaaactgtaagttaacttacctttgcttgtctaaacctaaaacaacaacaaaactacgaccacaagtacagttacatatttttgaaaattaaggttaagtgcagtgtaagtcaactatgcgaatggataacatgtttcaacatgaaactccgattgacgcatgtgcattctgaagagcggcgcggccgacgtctctcgaattgaagcaatgactcgcggaaccccgaaagcctttgggtggaaccctagggttccgcggaacacaggttgaagaacactg (SEQ ID NO: 6)pcDNA-DEST 40 gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagttaagctatcaacaagtttgtacaaaaaagctgaacgagaaacgtaaaatgatataaatatcaatatattaaattagattttgcataaaaaacagactacataatactgtaaaacacaacatatccagtcactatggcggccgcattaggcaccccaggctttacactttatgcttccggctcgtataatgtgtggattttgagttaggatccggcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtctgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaaagatctggatccggcttactaaaagccagataacagtatgcgtatttgcgcgctgatttttgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtgtgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggactggtgaaatgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggctccgttatacacagccagtctgcaggtcgaccatagtgactggatatgttgtgttttacagtattatgtagtctgttttttatgcaaaatctaatttaatatattgatatttatatcattttacgtttctcgttcagctttcttgtacaaagtggttgatctagagggcccgcggttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtaccggtcatcatcaccatcaccattgagtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgcca cctgacgtc(SEQ ID NO: 7) Fusion Transposaseatgctcgagatggatccctccgacgcttcgccggccgcgcaggtggatctacgcacgccontaining wild-typetcggctacagtcagcagcagcaagagaagatcaaaccgaaggtgcgttcgacagtggcTcBuster sequencegcagcaccacgaggcactggtgggccatgggtttacacacgcgcacatcgttgcgctc and TALE DNA-agccaacacccggcagcgttagggaccgtcgctgtcacgtatcagcacataatcacggbinding domaincgttgccagaggcgacacacgaagacatcgttggcgtcggcaaacagtggtccggcgctargeting humanacgcgccctggaggccttgttgactgatgctggtgagcttagaggacctcctttgcaa AAVS1cttgatacaggccagcttctgaaaatcgccaagaggggtggggtcaccgcggtcgaggccgtacacgcctggagaaatgcactgaccggggctcctcttaacCTGACCCCAGACCAGGTAGTCGCAATCGCGTCAAACGGAGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCCCACGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGTCGCATGACGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTGACGGCCAAGCTGGCCGGGGGCGCCCCCGCCGTGGGCGGGGGCCCCAAGGCCGCCGATAAATTCGCCGCCACCatgatgttgaattggctgaaaagtggaaagcttgaaagtcaatcacaggaacagagttcctgctaccttgagaactctaactgcctgccaccaacgctcgattctacagatattatcggtgaagagaacaaagctggtaccacctctcgcaagaagcggaaatatgacgaggactatctgaacttcggttttacatggactggcgacaaggatgagcccaacggactttgtgtgatttgcgagcaggtagtcaacaattcctcacttaacccggccaaactgaaacgccatttggacacaaagcatccgacgcttaaaggcaagagcgaatacttcaaaagaaaatgtaacgagctcaatcaaaagaagcatacttttgagcgatacgtaagggacgataacaagaacctcctgaaagcttcttatctcgtcagtttgagaatagctaaacagggcgaggcatataccatagcggagaagttgatcaagccttgcaccaaggatctgacaacttgcgtatttggagaaaaattcgcgagcaaagttgatctcgtccccctgtccgacacgactatttcaaggcgaatcgaagacatgagttacttctgtgaagccgtgctggtgaacaggttgaaaaatgctaaatgtgggtttacgctgcagatggacgagtcaacagatgttgccggtcttgcaatcctgcttgtgtttgttaggtacatacatgaaagctcttttgaggaggatatgttgttctgcaaagcacttcccactcagacgacaggggaggagattttcaatcttctcaatgcctatttcgaaaagcactccatcccatggaatctgtgttaccacatttgcacagacggtgccaaggcaatggtaggagttattaaaggagtcatagcgagaataaaaaaactcgtccctgatataaaagctagccactgttgcctgcatcgccacgctttggctgtaaagcgaataccgaatgcattgcacgaggtgctcaatgacgctgttaaaatgatcaacttcatcaagtctcggccgttgaatgcgcgcgtcttcgctttgctgtgtgacgatttggggagcctgcataaaaatcttcttcttcataccgaagtgaggtggctgtctagaggaaaggtgctgacccgattttgggaactgagagatgaaattagaattttcttcaacgaaagggaatttgccgggaaattgaacgacaccagttggttgcaaaatttggcatatatagctgacatattcagttatctgaatgaagttaatctttccctgcaagggccgaatagcacaatcttcaaggtaaatagccgcattaacagtattaaatcaaagttgaagttgtgggaagagtgtataacgaaaaataacactgagtgttttgcgaacctcaacgattttttggaaacttcaaacactgcgttggatccaaacctgaagtctaatattttggaacatctcaacggtcttaagaacacctttctggagtattttccacctacgtgtaataatatctcctgggtggagaatcctttcaatgaatgcggtaacgtcgatacactcccaataaaagagagggaacaattgattgacatacggactgatacgacattgaaatcttcattcgtgcctgatggtataggaccattctggatcaaactgatggacgaatttccagaaattagcaaacgagctgtcaaagagctcatgccatttgtaaccacttacctctgtgagaaatcattttccgtctatgtagccacaaaaacaaaatatcgaaatagacttgatgctgaagacgatatgcgactccaacttactactatccatccagacattgacaacctttgtaacaacaagcaggctcagaaatcccact ga(SEQ ID NO: 8) Flexible linker GGSGGSGGSGGSGTS (Example 4)(SEQ ID NO: 9) Flexible linkerGGAGGTAGTGGCGGTAGTGGGGGCTCCGGTGGGAGCGGCACCTCA (Example 4)(SEQ ID NO: 10) TALE domainatgctcgagatggatccctccgacgcttcgccggccgcgcaggtggatctacgcacgctargeting hAAVS1tcggctacagtcagcagcagcaagagaagatcaaaccgaaggtgcgttcgacagtggcsite (Example 5)gcagcaccacgaggcactggtgggccatgggtttacacacgcgcacatcgttgcgctcagccaacacccggcagcgttagggaccgtcgctgtcacgtatcagcacataatcacggcgttgccagaggcgacacacgaagacatcgttggcgtcggcaaacagtggtccggcgcacgcgccctggaggccttgttgactgatgctggtgagcttagaggacctcctttgcaacttgatacaggccagcttctgaaaatcgccaagaggggtggggtcaccgcggtcgaggccgtacacgcctggagaaatgcactgaccggggctcctcttaacCTGACCCCAGACCAGGTAGTCGCAATCGCGTCAAACGGAGGGGGAAAGCAAGCCCTGGAAACCGTGCAAAGGTTGTTGCCGGTCCTTTGTCAAGACCACGGCCTTACACCGGAGCAAGTCGTGGCCATTGCATCCCACGACGGTGGCAAACAGGCTCTTGAGACGGTTCAGAGACTTCTCCCAGTTCTCTGTCAAGCCCACGGGCTGACTCCCGATCAAGTTGTAGCGATTGCGTCGCATGACGGAGGGAAACAAGCATTGGAGACTGTCCAACGGCTCCTTCCCGTGTTGTGTCAAGCCCACGGTTTGACGCCTGCACAAGTGGTCGCCATCGCCTCCAATATTGGCGGTAAGCAGGCGCTGGAAACAGTACAGCGCCTGCTGCCTGTACTGTGCCAGGATCATGGACTGAC (SEQ ID NO: 11)Flexible linkerGGCCAAGCTGGCCGGGGGCGCCCCCGCCGTGGGCGGGGGCCCCAAGGCCGCCGATAAA (Example 5)TTCGCCGCCACC (SEQ ID NO: 12) Mutant TcBusterMMLNWLKSGKLESQSQEQSSCYLENSNCLPPTLDSTDIIGEENKAGTTSRKKRKYDED transposaseYLNFGFTWTGDKDEPNGLCVICEQVVNNSSLNPAKLKRHLDTKHPTLKGKSEYFKRKCcontaining V377T, NELNQKKHTFERYVRDDNKNLLKASYLVSLRIAKQGEAYTIAEKLIKPCTKDLTTCVF E469K, D189A,GEKFASKVDLVPLSATTISRRIEDMSYFCEAVLVNRLKNAKCGFTLQMDESTDVAGLAK573E and E578LILLVFVRYIHESSFEEDMLFCKALPTQTTGEEIFNLLNAYFEKHSIPWNLCYHICTDGAKAMVGVIKGVIARIKKLVPDIKASHCCLHRHALAVKRIPNALHEVLNDAVKMINFIKSRPLNARVFALLCDDLGSLHKNLLLHTETRWLSRGKVLTRFWELRDEIRIFFNEREFAGKLNDTSWLQNLAYIADIFSYLNEVNLSLQGPNSTIFKVNSRINSIKSKLKLWEECITKNNTKCFANLNDFLETSNTALDPNLKSNILEHLNGLKNTFLEYFPPTCNNISWVENPFNECGNVDTLPIKEREQLIDIRTDTTLKSSFVPDGIGPFWIKLMDEFPEISERAVKLLMPFVTTYLCEKSFSVYVATKTKYRNRLDAEDDMRLQLTTIHPDIDNLCNNKQAQKSH (SEQ ID NO: 13)

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A fusion transposase comprising a TcBustertransposase amino acid sequence and a DNA sequence specific bindingdomain, wherein the TcBuster transposase amino acid sequence has atleast 70% identity to full-length SEQ ID NO: 1 and an amino acidsubstitution of D189A, V377T, E469K, K573E, E578L, or any combinationthereof, when numbered in accordance with SEQ ID NO:
 1. 2. The fusiontransposase of claim 1, wherein the DNA sequence specific binding domaincomprises a TALE domain, zinc finger domain, AAV Rep DNA-binding domain,or any combination thereof.
 3. The fusion transposase of claim 1,wherein the DNA sequence specific binding domain comprises a TALEdomain.
 4. The fusion transposase of claim 1, wherein the TcBustertransposase amino acid sequence has at least 80%, at least 90%, at least95%, at least 98%, or at least 99% identity to full-length SEQ ID NO: 1.5. The fusion transposase of claim 1, wherein the TcBuster transposaseamino acid sequence has increased transposition efficiency in comparisonto a wild-type TcBuster transposase having amino acid sequence SEQ IDNO: 1 when measured by an assay that comprises: (i) introducing thefusion transposase or the wild-type TcBuster transposase and a TcBustertransposon containing a reporter cargo cassette into a population ofcells and (ii) detecting transposition of the reporter cargo cassette ingenome of the population of cells induced by the fusion transposase andthe wild-type TcBuster transposase, respectively.
 6. The fusiontransposase of claim 1, wherein the TcBuster transposase amino acidsequence and the DNA sequence specific binding domain are separated by alinker.
 7. The fusion transposase of claim 6, wherein the linkercomprises at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 15, at least 20, or at least50 amino acids.
 8. The fusion transposase of claim 6, wherein the linkercomprises SEQ ID NO:
 9. 9. The fusion transposase of claim 1, whichcomprises amino acid substitutions of D189A, V377T, and E469K, whennumbered in accordance with SEQ ID NO:
 1. 10. The fusion transposase ofclaim 9, which further comprises an amino acid substitution K573E and/orE578L, when numbered in accordance with SEQ ID NO:
 1. 11. The fusiontransposase of claim 10, which comprises amino acid substitutions ofD189A, V377T, E469K, K573E, and E578L when numbered in accordance withSEQ ID NO:
 1. 12. A polynucleotide that codes for the fusion transposaseof claim
 1. 13. The polynucleotide of claim 12, wherein thepolynucleotide comprises a messenger RNA (mRNA) that encodes the fusiontransposase.
 14. The polynucleotide of claim 13, wherein the mRNAencoding the fusion transposase is chemically modified.
 15. A system forgenome editing comprising: a transposon and a fusion transposase or apolynucleotide coding for the fusion transposase, wherein the fusiontransposase comprises a TcBuster transposase amino acid sequence and aDNA sequence specific binding domain, wherein the TcBuster transposaseamino acid sequence (i) recognizes the transposon, (ii) has at least 70%identity to full-length SEQ ID NO: 1, and (iii) comprises an amino acidsubstitution of D189A, V377T, E469K, K573E, E578L, or any combinationthereof, when numbered in accordance with SEQ ID NO:
 1. 16. The systemof claim 15, wherein the transposon is present in a mini-circle plasmid.17. The system of claim 15, wherein the transposon comprises a cargocassette positioned between two inverted repeats.
 18. The system ofclaim 17, wherein a left inverted repeat of the two inverted repeatscomprises an amino acid sequence having at least 80% identity to SEQ IDNO: 3, or a right inverted repeat of the two inverted repeats comprisesan amino acid sequence having at least 80% identity to SEQ ID NO:
 4. 19.The system of claim 17, wherein the cargo cassette is in a reversedirection.
 20. The system of claim 17, wherein the cargo cassettecomprises a transgene coding for a cellular receptor selected from thegroup consisting of: a T cell receptor (TCR), a B cell receptor (BCR), achimeric antigen receptor (CAR), and any combination thereof.
 21. Thesystem of claim 15, wherein the fusion transposase comprises amino acidsubstitutions of D189A, V377T, and E469K, when numbered in accordancewith SEQ ID NO:
 1. 22. The system of claim 21, wherein the fusiontransposase further comprises an amino acid substitution K573E and/orE578L, when numbered in accordance with SEQ ID NO:
 1. 23. The system ofclaim 22, wherein the fusion transposase comprises amino acidsubstitutions of D189A, V377T, E469K, K573E, and E578L when numbered inaccordance with SEQ ID NO:
 1. 24. A method of genome editing comprising:introducing into a cell a transposon and a fusion transposase or apolynucleotide encoding the fusion transposase, wherein the fusiontransposase comprises a TcBuster transposase amino acid sequence and aDNA sequence specific binding domain, wherein the TcBuster transposaseamino acid sequence (i) recognizes the transposon, (ii) has at least 70%identity to full-length SEQ ID NO: 1, and (iii) comprises an amino acidsubstitution of D189A, V377T, E469K, K573E, E578L, or any combinationthereof, when numbered in accordance with SEQ ID NO: 1.